Live Pups Research Articles

Efficient breeding system of infertile Niemann-Pick disease type C model mice by in vitro fertilization and embryo transfer.

Niemann-Pick disease type C (NPC) is a lethal genetic disease with mutations in NPC1 or NPC2 gene. Npc1-deficient (Npc1-/-) mice have been used as a model for NPC pathogenesis to develop novel therapies for NPC. However, Npc1-/- mice are infertile; thus, securing sufficient numbers for translational research is difficult. Hence, we attempted reproductive engineering techniques such as in vitro fertilization (IVF) and sperm cryopreservation. For the first time, we succeeded in producing fertilized oocytes via IVF using male and female Npc1-/- mice. Fertilized oocytes were also obtained via IVF using cryopreserved sperm from Npc1-/- mice. The obtained fertilized oocytes normally developed into live pups via embryo transfer, and they eventually exhibited NPC pathogenesis. These findings are useful for generating an efficient breeding system that overcomes the reproductive challenges of Npc1-/- mice and will contribute to developing novel therapeutic methods using NPC model mice.

O-185 Far away so close: remote ICSI using an AI-powered robot

Abstract Study question Can intracytoplasmic sperm injection (ICSI) be performed remotely using a personal computer and an intelligent robotic micromanipulation system? Summary answer An AI-enhanced robotic micromanipulation system was developed that allows successful remote execution of ICSI by experts or non-experts. What is known already ICSI has become the method of choice for fertilization of eggs during IVF, but it remains technically demanding, requiring manual control of highly specialized equipment by expert embryologists. Limited automation of the procedure has been achieved but most steps are still performed manually. Remote performance of ICSI has never been attempted. Study design, size, duration To achieve automated ICSI, we explored individual commands for each step, issued from an offsite computer. Steps included AI-assisted sperm selection, laser immobilization, random oocyte positioning, opening a path in the zona pellucida prior to needle entry, and Piezo-mediated oolemma breakage. The modified protocols were optimized using mouse oocytes and mouse sperm and then tested using the automated system to inject hamster oocytes with human sperm. Finally, the system was evaluated in consenting patients. Participants/materials, setting, methods The system consists of an inverted microscope, a motorized stage, motorized micromanipulators, laser, piezo-electric actuator, and microinjectors, all controlled by proprietary digital and AI-driven software. Development of this system was a collaborative effort across four countries in three laboratories, including an engineering and embryo research facility, an experimental embryology laboratory, and an IVF clinic. The pilot clinical procedures were performed after full IRB review and trial registration. Main results and the role of chance To assess the safety of using a laser on sperm tails for immobilization, manual ICSI was performed on mouse oocytes with mouse sperm immobilized using different laser settings. Once the method was optimized, fertilization (n = 176, 98.8%) and blastocyst formation (80.7%) in the experimental group were similar to those in the controls without laser (n = 173, 99.4% and 80.6%, respectively) (p > 0.05). Subsequently, 90 blastocysts from the experimental group were transferred to pseudopregnant mice resulting in 40.4% live pups. This was comparable to the results with control embryos (n = 90, 47.8%) (p = 0.098). The remotely controlled robotic system was then tested in hamster oocytes, demonstrating survival (n = 102, 94.1%) similar to the manually injected control group (n = 102, 97.1%) (p = 0.3). In the clinical pilot trial in four patients, 22 oocytes were assigned to remote ICSI, achieving survival (95%), fertilization (81%), and blastocyst formation (65%) comparable to manually performed ICSI controls on sibling oocytes. These results are within the benchmark values established in the Vienna consensus. Throughout all experiments, the system allowed users to successfully perform ICSI remotely issuing commands by pressing single buttons on a computer screen from various locations, ranging in distance from a few meters to over 9,000 kilometers away from the laboratory. Limitations, reasons for caution This experimental ICSI system requires operators in the laboratory to insert and position the microtools and handle the eggs and sperm before and after ICSI. The system also requires a broadband internet connection with at least 50MBPs speed in upload and download. Latency or delayed reaction is a rare possibility. Wider implications of the findings The functionality of a remotely controlled automated ICSI system was validated. The system demonstrates efficiency gains, even with remote operation. The procedures can be programmed to achieve full microscopic ICSI using a single command. This system represents a significant advancement in automation of IVF. Trial registration number Clinicaltrials.gov: NCT06074835 IRB approval: RA-2023-01 IRB ID: CONBIOÉTICA-09-CEI-001-20170131

P-316 Fertility, pregnancy outcomes, and perinatal results related to experimental endometriosis in a murine model

Abstract Study question Does the transplantation of endometrial tissue fragments into the peritoneal wall of female mice reduce natural fertility and lead to compromised obstetric and perinatal outcomes? Summary answer Experimental murine endometriosis did not compromise fertility, but females with more ectopic endometria had litters with reduced birth weight and fewer offspring at weaning. What is known already It has been demonstrated that endometriosis is strongly associated with subfertility and is increasingly linked to late pregnancy complications, such as those caused by placental insufficiency. At the molecular level, the presence of ectopic endometrium may perturb the endometrial hormonal, cellular, and immunological milieu, negatively influencing the decidualization, placentation, and developmental programming of the embryo. However, it remains unclear if and how such early aberrant reproductive development relates to pregnancy outcomes in endometriosis. Thus, the use of animal models may be very useful in deepening our understanding of the pathogenic mechanisms of infertility and compromised pregnancy outcomes related to endometriosis. Study design, size, duration C57BL/6J female mice were divided into three groups: Control (42 animals underwent sham surgery), Endometriosis1 (19 animals underwent surgery for transplantation of one fragment of endometrial tissue on each side of the peritoneal wall), and Endometriosis2 (23 animals received two fragments on each side). The maximum experiment duration was 15 weeks, including a 4-week rest period post-surgery, up to 4 weeks for mating, 3 weeks for delivery, and 4 weeks until weaning of the offspring. Participants/materials, setting, methods Reproductive-age female mice from the three groups had a 4-week rest period post-surgery. Then, they were paired for mating with proven fertile males and checked for the presence of a vaginal plug daily. Pregnant females were euthanized after weaning the litter at 4 weeks. Tissue from the implants was collected for histological analysis. The occurrence of pregnancy, gestation duration, litter size, pup weight at birth and weaning, and mating return were compared among the groups. Main results and the role of chance All females in the experimental groups were confirmed for the presence of ectopic endometrium through histology. No statistically significant differences were observed in pregnancy rates, gestation duration, litter size at birth, and the number of live pups at birth when comparing the three groups (C, E1, and E2). However, the litter weight at birth was significantly lower in group E2 (Medial (Md): 8g; Percentile (P)25: 6; P75: 10) than in groups C (Md: 10g; P25: 8; P75: 10) and E1 (Md: 10g; P25: 10; P75: 12), and significantly higher in E1 compared to C. The number of live pups at weaning was significantly lower in E2 (Md: 5; P25: 3.5; P75: 7) compared to groups C (Md: 7; P25: 5; P75: 7) and E1 (Md: 8; P25: 7; P75: 8), and significantly higher in E1 than in C. The litter weight at weaning was significantly lower in E2 (Md: 96g; P25: 67; P75: 109) than in both E1 (Md: 116g; P25: 110; P75: 128) and C (Md: 104g; P25: 96; P75: 112). The litter weight at weaning was higher in E1 compared to group C. There was no statistically significant difference in mating return rates among the three groups. Limitations, reasons for caution This study is limited by the use of an animal model that does not have spontaneous endometriosis. Wider implications of the findings Litters from the E2 group may have had impaired development during gestation, as indicated by lower birth weight and higher perinatal mortality. Although this is not an ideal translational model for studying endometriosis-related infertility, it may serve as a suitable model for evaluating gestational and perinatal outcomes associated with endometriosis. Trial registration number Not applicable

Disruption of the SP-A/SP-R210L (MYO18Aα) pathway prolongs gestation and reduces fetal survival during lipopolysaccharide-induced parturition in late gestation.

Prolonged labor can lead to infection, fetal distress, asphyxia, and life-threatening harm to both the mother and the baby. Surfactant protein A (SP-A) was shown to contribute to the maintenance of pregnancy and timing of term labor. SP-A modulates the stoichiometric expression of the SP-R210L and SP-R210S isoforms of the SP-R210 receptor on alveolar macrophages (AMs). Lack of SP-R210L dysregulates macrophage inflammatory responses. We asked whether SP-A alters normal and inflammation-induced parturition through SP-R210 using SP-A- and SP-R210L-deficient mice. Labor and delivery of time-pregnant mice were monitored in real time using a time-lapse infrared camera. Intrauterine injection with either vehicle or Escherichia coli lipopolysaccharide (LPS) on embryonic (E) day 18.5 post coitus was used to assess the effect of gene disruption in chorioamnionitis-induced labor. We report that either lack of SP-A or disruption of SP-R210L delays parturition by 0.40 and 0.55 days compared with controls, respectively. LPS induced labor at 0.60, 1.01, 0.40, 1.00, and 1.31 days earlier than PBS controls in wild type (WT), SP-A-deficient, littermate controls, heterozygous, and homozygous SP-R210L-deficient mice, respectively. Lack of SP-A reduced litter size in PBS-treated mice, whereas the total number of pups delivered was similar in all LPS-treated mice. The number of live pups, however, was significantly reduced by 50%-70% in SP-A and SP-R210L-deficient mice compared with controls. Differences in gestational length were not associated with intrauterine growth restriction. The present findings support the novel concept that the SP-A/SP-R210 pathway modulates timely labor and delivery and supports fetal lung barrier integrity during fetal-to-neonatal transition in term pregnancy.NEW & NOTEWORTHY To our knowledge, this study is the first to report that SP-A prevents delay of labor and inflammation-induced stillbirth through the receptor SP-R210L.

Inhibition of Caspase 1 Reduces Blood Pressure, Cytotoxic NK Cells, and Inflammatory T-Helper 17 Cells in Placental Ischemic Rats.

Preeclampsia (PE) is characterized by maternal hypertension, fetal growth restriction (FGR), and increased inflammation and populations of cytotoxic NK cells (cNKs) and inflammatory T-Helper 17 cells (TH17s). Both cytotoxic NK cells and TH17 cells are heavily influenced via IL-1β signaling. Caspase 1 activity leads to the release of the inflammatory cytokine IL-1β, which is increased in women with PE. Therefore, we tested the hypothesis that the inhibition of Caspase 1 with VX-765 in rats with reduced uterine perfusion pressure (RUPP) will attenuate PE pathophysiology. On gestation day (GD) 14, timed pregnant Sprague-Dawley rats underwent the RUPP or Sham procedure and were separated into groups that received either vehicle or VX-765 (50 mg/kg/day i.p.). On GD19, MAP was measured via carotid catheter and blood and tissues were collected. Bio-Plex and flow cytometry analysis were performed on placental tissues. Placental IL-1β was increased in the RUPP rats vs. the Sham rats and treatment with VX-765 reduced IL-1β in the RUPP rats. Caspase 1 inhibition reduced placental cNKs and TH17s in RUPP rats compared to vehicle-treated RUPP rats. Increased MAP was observed in RUPP rats compared with Sham rats and was reduced in RUPP + VX-765 rats. Placental reactive oxygen species (ROS) were elevated in RUPP rats compared to Sham rats. VX-765 administration reduced ROS in treated RUPP rats. Caspase 1 inhibition increased the number of live pups, yet had no effect on fetal weight or placental efficiency in the treated groups. In conclusion, Caspase 1 inhibition reduces placental IL-1β, inflammatory TH17 and cNK populations, and reduces MAP in RUPP rats. These data suggest that Caspase 1 is a key contributor to PE pathophysiology. This warrants further investigation of Caspase 1 as a potential therapeutic target to improve maternal outcomes in PE.

Time elapsed between ovulation and insemination determines the quality of fertilized rat oocytes.

Genetically modified rats are valuable models in human disease research. We recently developed an improved system for rat sperm cryopreservation and in vitro fertilization (IVF) that facilitates the efficient production and preservation of genetically modified rats. In the IVF procedure performed using frozen-thawed rat sperm, the IVF schedule is fixed to ensure timely hormone administration and oocyte collection. To enhance the flexibility of the IVF schedule, possible periods of postovulated rat oocytes with normal fertility and developmental abilities should be determined. Therefore, in this study, we examined the fertilization and developmental ability of incubated oocytes 1-13 h after oocyte collection at 9:00 AM. The fertilization rate decreased 7 h after oocyte collection, and abnormally fertilized oocytes appeared 10 h after oocyte collection. The developmental rate also decreased 7 h after oocyte collection; however, live pups were obtained from oocytes 12 h after oocyte collection. In summary, ovulated rat oocytes exhibited a high developmental ability after IVF for up to 4 h after oocyte collection.

The adverse effects of vitrification on mouse embryo development and metabolic phenotype in offspring.

Embryo vitrification is a standard procedure in assisted reproductive technology. Previous studies have shown that frozen embryo transfer is associated with an elevated risk of adverse maternal and neonatal outcomes. This study aimed to explore the effects of mouse blastocyst vitrification on the phenotype of vitrified-warmed blastocysts, their intrauterine and postnatal development, and the long-term metabolic health of the derived offspring. The vitrified-warmed blastocysts (IVF + VT group) exhibited reduced mitochondrial activity, increased apoptotic levels, and decreased cell numbers when compared to the fresh blastocysts (IVF group). Implantation rates, live pup rates, and crown-rump length at E18.5 were not different between the two groups. However, there was a significant decrease in fetal weight and fetal/placental weight ratio in the IVF + VT group. Furthermore, the offspring of the IVF + VT group at an age of 36 weeks had reduced whole energy consumption, impaired glucose and lipid metabolism when compared with the IVF group. Notably, RNA-seq results unveiled disturbed hepatic gene expression in the offspring from vitrified-warmed blastocysts. This study revealed the short-term negative impacts of vitrification on embryo and fetal development and the long-term influence on glucose and lipid metabolism that persist from the prenatal stage into adulthood in mice.

Cochlear pathology in preeclamptic rats: protective effects of vitamin D and magnesium sulfate.

This study investigated the possible degeneration in cochlear morphology induced by preeclampsia (PE) and the therapeutic/preventive effect of vitamin D (Vit D) and magnesium sulfate (MgSO4) used separately and together on feto-maternal outcomes. We created PE in rats using a reduced uterine perfusion pressure (RUPP) animal model and recorded blood pressure (BP), embryonic survival (ES), and embryonic weight (EW) and evaluated cochlear morphology by electron microscopy. The PE group had elevated BP, a decreased number and weight of live pups, and significant degeneration in the cochlea compared to the sham group. In the PEV group, we observed significant beneficial effects of Vit D supplementation at 14.5 and 19.5 dpc in terms of BP (p < 0.05), EW (p < 0.001), and cochlear degeneration compared to the PE group. In the PEM group, BP (p < 0.05) and cochlear degeneration nearly reached the level found in the sham group. However, although the EW was statistically different in the PE group, it did not reach sham group levels. We also observed that BP returned to sham level (p < 0.01) and noticed significant increases in the EW (p < 0.0001) and ES (p = 0.017) in the PEMV group compared to the PE group. According to the scanning electron microscope results, combined administration of VitD and MgSO4 is more effective than separate administration in improving cochlear degeneration induced by PE. The administration of Vit D and MgSO4 during pregnancy has beneficial effects on PE pathology and may play a significant role in preventing PE-related complications, including cochlear degeneration.

#208 : The Adverse Effects of Vitrification on Embryo Development and Metabolic Health in Mice Offspring

Background and Aims: Embryo vitrification is widely applied as a routine procedure of assisted reproductive technology. Cohort studies showed frozen embryo transfer (FET) was associated with an elevated risk of adverse maternal and neonatal outcomes compared with fresh embryo transfer. This study aimed to explore the effects of blastocyst vitrification on embryo development and long-term metabolic health in mice. Method: Mitochondrial function, apoptotic levels and cell numbers were examined in blastocysts with/without vitrification. Implantation rates, fetal growth and placentation were analysed after fresh and frozen blastocyst transfer. Energy consumptions were measured by indirect calorimetry. Metabolic tests were performed in adult mice offspring, including glucose tolerance, insulin tolerance and pyruvate tolerance. Serum lipid profiles were examined, and fetal and adult hepatic transcriptome status were detected by RNA-sequencing and qRT-PCR. Results: Mitochondrial dysfunction increased apoptotic levels and decreased cell numbers were observed in vitrified blastocysts. The implantation and live pup rates on embryonic day18.5 were not significantly different between the fresh and frozen groups. However, fetal weights and placental efficiencies such as the ratio of labyrinth layer/ junctional zone and microvascular density were significantly reduced after FET. Offspring aged 36 weeks in the FET group had decreased whole energy consumption, impaired glucose and pyruvate tolerance, insulin resistance, higher serum triglyceride, low-density lipoprotein and total cholesterol levels compared with the fresh group. However, these metabolic effects were not observed in mice offspring aged 14 weeks. The FET group also had altered hepatic gene expression and signaling pathways related to metabolic regulation, apoptotic cell clearance, and regulation of response to reactive oxygen species etc. Conclusion: The data indicated that blastocyst vitrification has short-term and long-term adverse effects in the mouse model. Further studies are needed to optimize vitrification procedure.

Characterization of fetal microchimeric immune cells in mouse maternal hearts during physiologic and pathologic pregnancies.

Introduction: During pregnancy, fetal cells can be incorporated into maternal tissues (fetal microchimerism), where they can persist postpartum. Whether these fetal cells are beneficial or detrimental to maternal health is unknown. This study aimed to characterize fetal microchimeric immune cells in the maternal heart during pregnancy and postpartum, and to identify differences in these fetal microchimeric subpopulations between normal and pregnancies complicated by spontaneous preterm induced by ascending infection. Methods: A Cre reporter mouse model, which when mated with wild-type C57BL/6J females resulted in cells and tissues of progeny expressing red fluorescent protein tandem dimer Tomato (mT+), was used to detect fetal microchimeric cells. On embryonic day (E)15, 104 colony-forming units (CFU) E. coli was administered intravaginally to mimic ascending infection, with delivery on or before E18.5 considered as preterm delivery. A subset of pregnant mice was sacrificed at E16 and postpartum day 28 to harvest maternal hearts. Heart tissues were processed for immunofluorescence microscopy and high-dimensional mass cytometry by time-of-flight (CyTOF) using an antibody panel of immune cell markers. Changes in cardiac physiologic parameters were measured up to 60days postpartum via two-dimensional echocardiography. Results: Intravaginal E. coli administration resulted in preterm delivery of live pups in 70% of the cases. mT + expressing cells were detected in maternal uterus and heart, implying that fetal cells can migrate to different maternal compartments. During ascending infection, more fetal antigen-presenting cells (APCs) and less fetal hematopoietic stem cells (HSCs) and fetal double-positive (DP) thymocytes were observed in maternal hearts at E16 compared to normal pregnancy. These HSCs were cleared while DP thymocytes persisted 28days postpartum following an ascending infection. No significant changes in cardiac physiologic parameters were observed postpartum except a trend in lowering the ejection fraction rate in preterm delivered mothers. Conclusion: Both normal pregnancy and ascending infection revealed distinct compositions of fetal microchimeric immune cells within the maternal heart, which could potentially influence the maternal cardiac microenvironment via (1) modulation of cardiac reverse modeling processes by fetal stem cells, and (2) differential responses to recognition of fetal APCs by maternal T cells.

Extracellular Vesicles-mediated recombinant IL-10 protects against ascending infection-associated preterm birth by reducing fetal inflammatory response.

Fetal inflammatory response mediated by the influx of immune cells and activation of pro-inflammatory transcription factor NF-κB in feto-maternal uterine tissues is the major determinant of infection-associated preterm birth (PTB, live births < 37 weeks of gestation). To reduce the incidence of PTB by minimizing inflammation, extracellular vesicles (EVs) were electroporetically engineered to contain anti-inflammatory cytokine interleukin (IL)-10 (eIL-10), and their efficacy was tested in an ascending model of infection (vaginal administration of E. coli) induced PTB in mouse models. EVs (size: 30-170 nm) derived from HEK293T cells were electroporated with recombinant IL-10 at 500 volts and 125 Ω, and 6 pulses to generate eIL-10. eIL-10 structural characters (electron microscopy, nanoparticle tracking analysis, ExoView [size and cargo content] and functional properties (co-treatment of macrophage cells with LPS and eIL-10) were assessed. To test efficacy, CD1 mice were vaginally inoculated with E. coli (1010CFU) and subsequently treated with either PBS, eIL-10 (500ng) or Gentamicin (10mg/kg) or a combination of eIL-10+gentamicin. Fetal inflammatory response in maternal and fetal tissues after the infection or treatment were conducted by suspension Cytometer Time of Flight (CyTOF) using a transgenic mouse model that express red fluorescent TdTomato (mT+) in fetal cells. Engineered EVs were structurally and functionally stable and showed reduced proinflammatory cytokine production from LPS challenged macrophage cells in vitro. Maternal administration of eIL-10 (10 µg/kg body weight) crossed feto-maternal barriers to delay E. coli-induced PTB to deliver live pups at term. Delay in PTB was associated with reduced feto-maternal uterine inflammation (immune cell infiltration and histologic chorioamnionitis, NF-κB activation, and proinflammatory cytokine production). eIL-10 administration was safe, stable, specific, delayed PTB by over 72 hrs and delivered live pups. The delivery of drugs using EVs overcomes the limitations of in-utero fetal interventions. Protecting IL-10 in EVs eliminates the need for the amniotic administration of recombinant IL-10 for its efficacy.

Activated platelet-rich plasma accelerate endometrial regeneration and improve pregnancy outcomes in murine model of disturbed endometrium.

Platelet Rich Plasma (PRP) contains high concentrations of growth factors, therefore, PRP activation results in their release, stimulating the process of healing and regeneration. The study was conducted to check whether activated platelet-rich plasma (aPRP) treatment can improve regeneration of the endometriumin an experimental model of ethanol-induced disturbed endometrium. Seventy-two female Wistar rats were randomly assigned into the control group, disturbed endometrium (DE) group and aPRP treated group. Activation of PRP was performed by adding thrombin. All the animals were sacrificed on day 1, day 3, day 6 and day 9 and samples were taken from the miduterine horn. Quantification of Cytokine and chemokine profiles of activated and non-activated PRP for CCL2, TNF- α, IL-1β, CXCL8, CXCL10, IL2, IL4, IL-6 IL-10, IL-12, IL-17A, TGF- β, IFN-γ was carried out. Functional and structural recovery of the endometrium was analyzed by hematoxylin-eosin (HE) and immunohistochemical (IHC) analyses. HE confirmed proliferated epithelial lining and stromal reconstruction with decreased fibrosis in PRP treated group compared to the DE group. Epithelial thickness in aPRP treated on day 1, day 3, day 6 and day 9 revealed an significant increase (p ≤ 0.05). Significantly stronger IHC expression of alpha smooth muscle actin, Cytokeratin 18, Cytokeratin 19, Connexin-40, E-Cadherin, Claudin-1, Zona Occludin-1was found in the aPRP treated group compared to the DE group. Furthermore, aPRP treatment was associated with birth of live pups. Our results suggest that intrauterine administration of aPRP stimulated and accelerated the regeneration of endometrium in the murine model of disturbed endometrium.

O-090 Endocrine disruptors

Abstract Female mammals, including women, are exposed to endocrine disrupting chemicals (EDCs) on a daily basis. This is of concern because many EDCs inhibit ovarian follicle growth and steroidogenesis, processes that are critical for maintaining female fertility. Our research focuses on the effects of EDCs known as phthalates on female fertility. Phthalates are of interest because they are ubiquitous synthetic chemicals used as plasticizers and stabilizers in a myriad of consumer products, including everything from shower curtains to children’s toys to cosmetics and personal care products such as perfumes, nail polish, deodorants, and lotions. Phthalates are also used in pesticides, wood finishes, adhesives, solvents, lubricants, defoaming agents, and in medical devices including tubing, blood bags, surgical gloves, and dialysis equipment. Despite the widespread use of phthalates and ubiquitous human exposure, limited information is available about the effects of environmentally relevant phthalate mixtures on female reproduction. Thus, we tested the hypothesis that prenatal exposure to an environmentally relevant phthalate mixture affects female reproduction in the F1, F2, and F3 generations of mice. To test this hypothesis, pregnant CD-1 mice were orally dosed with vehicle control (corn oil) or a phthalate mixture (20µg/kg/day-500mg/kg/day) daily from gestational day 10 to birth. The mixture was based on urinary phthalate metabolite levels measured pregnant women in the United States, and consisted of 35% diethyl phthalate, 21% di(2-ethylhexyl) phthalate, 15% dibutyl phthalate, 15% di-isononyl phthalate, 8% diisobutyl phthalate, and 5% benzylbutyl phthalate. Adult F1 females born to these dams were used to create the F2 generation by mating them with unexposed males and F2 females were used to create the F3 generation by mating them with unexposed males. At selected times, estrous cyclicity and fertility indices were monitored and ovaries and sera were collected for analysis. Our data indicate that prenatal exposure to the phthalate mixture significantly decreases anogenital distance, testosterone levels, and litter size, but it significantly increases abnormal estrous cyclicity, uterine weight, the number of cystic ovaries, follicle-stimulating hormone levels, and luteinizing hormone levels compared to control in the F1 generation. Further, our data indicate that prenatal exposure to the phthalate mixture increases body weight, uterine weight, the number of cystic ovaries, and time to pregnancy and it decreases testosterone levels compared to control in the F2 generation. In addition, our data indicate that prenatal exposure to the phthalate mixture increases body weight, uterine weight, the number of cystic ovaries, and abnormal cyclicity and it decreases anogenital distance and the number of live pups compared to control in the F3 generation. Collectively, these data suggest that prenatal exposure to an environmentally relevant mixture of phthalates adversely affects reproductive function in a multi- and transgenerational manner in female mice. These findings have increased our understanding of the mechanisms by which EDCs cause female reproductive toxicity. This information eventually may lead to the development of novel targets for the prevention or treatment of infertility induced by EDCs.

Toxic effect of Syzygium guineense ethanolic extract on female reproduction in rats: An evidence from a 10 week repeated-dose toxicity study

IntroductionIt has been reported that some herbal products affect reproduction. To date, reproductive toxicity of Syzygium guineense has not been investigated although the plant is widely used in treating fertility related problems. Thus, the objective of the current study was to investigate the toxic effects of 70% ethanol extract of S. guineense leaves on the reproductive function and histopathology of reproductive organs in female rats. MethodsEighty female Wistar albino rats were randomly divided into four groups where each group consisted of 20 rats. Rats in the first three groups were treated with S. guineense extract at doses of 250, 500 and 1000 mg/kg body weight, respectively. The fourth group served as a control group. The rats were treated for ten consecutive weeks. The length of estrous cycle, reproductive indices, pregnancy outcomes, and number of postnatal deaths were recorded. At necropsy, organ weight was measured, gross and histopathological examinations of ovaries, uterus, and vagina were conducted. ResultsTreatment of rats, with high dose (1000 mg/kg) of S. guineense, significantly prolonged the duration of estrous cycle and reduced weight of uterus and ovaries as well as the number of total and live birth pups. However, there were no significant changes observed in reproductive indices and gross morphology as well as histopathology of ovaries, uterus, and vagina. ConclusionAdministration of high doses of S. guineense could be toxic to some aspects of the reproductive system of female rats and might also affect reproduction. Therefore, consuming high dose of S. guineense leaves is not recommended.

Pregnancy and spontaneous partial abortion associated with salmonella in a dog

A 2-year mixed breed dog was presented on day 50 after breeding for an emergency condition (greenish brown vulvar discharge and abortion). The patient was underweight (lower body condition score), had nonfetid vulvar discharge, and was negative for Ferguson reflux. Patient had 10 premature fetuses (determined via transabdominal ultrasonography and radiography). Serum progesterone concentration was 1.25 ng/ml. Overnight hospitalization therapies included intravenous fluids, terbutaline, fenbendazole, altrenogest, progesterone, and antibiotics. Based on vaginal discharge culture results (salmonella), antibiotics treatment was altered. Three days later, neutrophilia with left shift and a fetal death were observed. Because cesarean surgery with ovariohysterectomy was declined by the owner, altrenogest and terbutaline treatments were discontinued to permit whelping. Patient was in dystocia after delivering 2 stillborn and 2 live pups. One pup was delivered medically and 1 necrotic pup via cesarean surgery. Intrauterine culture (collected during surgery) was negative. On day 7 postpartum, the patient and 3 remaining pups were healthy. This case demonstrated the difficulties of managing fetal-placental infections.

A preliminary study on placental damage associated to experimental neosporosis in BALB/c mice.

Neospora caninum is a protozoan parasite which can infect a range of animals, including dogs, cattle, and sheep. Bovine neosporosis, which mainly causes abortion in cattle, results in substantial economic losses worldwide. To study the effects of N. caninum infection on the placenta, a pregnant mouse model for N. caninum infection was established. The litter size (8.6 ± 1.5) and the number of live pups (6.4 ± 1.8) of infected dams were significantly lower compared with those of non-infected dams. Trophoblast cell shrinkage and a large number of apoptosomes were detected in the placentas of the infected group. The parasite load in the placental tissue was significantly higher with time after infection. Likewise, apoptosis of placental trophoblast cells significantly increased with time after infection. Among the 66 apoptotic genes detected in this study, eight genes, including Bcl-2, were significantly differentially expressed by about > tenfold in infected and uninfected mice. The expression of BAX and tumor necrosis factor-alpha (TNF-α) was upregulated in the placental cells of the infected mice, whereas the expression of BCL-2 was downregulated. Enzyme-linked immunosorbent assays (ELISAs) showed that apoptotic protease caspase-3 level was significantly increased in placental cell suspension, and insulin-like growth factor (IGF)-2 level was significantly reduced. Acetylcholine (ACH) and placental prolactin (PL) levels were initially decreased but eventually increased. In summary, infection of mice with N. caninum caused apoptotic damage to the placental tissues, cells, and genes and affected the normal physiological functions of placenta, which may largely explain the adverse pregnancy outcomes caused by N. caninum infection in mice.

Prenatal zestoretic exposure ameliorates developmental landmarks in rat offspring

Zestoretic, a combination of lisinopril and hydrochlorothiozide used as anti-hypertension drug prescribed even during pregnancy. Role of zestoretic on the developmental landmars of young ones exposed prenatally are yet to be established. Hence, the present investigation initiated to test in Wistar rats. Three groups of inseminated female rats were oral gavaged with zestoretic (lisinopril varied concentrations + hydrochlorothiozide 12.5mg) 25, 50 and 100 mg/Kg body weight (BW) on gestation days 7, 9, 11 and 13, whereas control group with double distilled water. Clinical toxicity found in few dams from 50 and 100 mg/Kg BW received rat shown aggressive behavior during experimentation. The developmental landmarks, such as pinna attachment, ear opening, fur development, eye opening, upper and lower incisor eruption, crown rump length, vaginal opening and testes descend were measured periodically on their postnatal days in all the young ones. The number of live pups in zestoretic 50, 100 mg/Kg BW administered females are significantly less (P

KSOM-R supports both mouse and rat preimplantation embryo development in vitro

A modified KSOM for rat embryo culture (KSOM-R), which has enriched taurine, glycine, glutamic acid, and alanine, promoted rat embryo development in vitro. Since mice and rats share similar amino acid profiles in their female reproductive tracts, this study explored whether KSOM-R would also have a positive effect on mouse embryo development and if KSOM-R modifications could extend its shelf time at 2–8 °C for consistency. We first examined the effects of newly made (≤1 month at 2–8 °C) antibiotics-free KSOM-R (mKSOM-R), antibiotics-free KSOM (mKSOM) and KSOM on the development of in vivo or in vitro derived C57BL/6NJ zygotes. We then investigated the effect of extended shelf life (6 months at 2–8 °C) of mKSOM-R and mKOSM on the development of C57BL/6NJ mouse and Sprague Dawley (SD) rat embryos. The results showed that there were no significant differences in cleavage, blastocyst, and hatching rates of C57BL/6NJ embryos among the three freshly made media. After 6 months of storage at 2–8 °C, mKSOM-R and mKSOM were still able to support the development of in vivo C57BL/6NJ zygotes at comparable rates seen with newly made (≤1 month at 2–8 °C) KSOM (control) in terms of cleavage, blastocyst formation and hatching. There were also no significant differences in total cell numbers in day 4 blastocysts among the three groups. After surgical embryo transfers, C57BL/6NJ blastocysts cultured in mKSOM-R (6 months at 2–8 °C) and newly made (≤1 month at 2–8 °C) KSOM culture developed into live pups. These pups had no gross abnormalities in animal morphology and growth. SD zygotes cultured in mKSOM-R stored at 2–8 °C for 6 months developed at comparable rates in cleavage, blastocyst and hatching rates when compared to those cultured in newly made mKSOM-R (≤1 month at 2–8 °C). The data showed that, although no significant beneficial effects were observed on mouse embryo development, mKSOM-R was able to support both mouse and rat embryo development in vitro. Additionally, mKSOM-R and mKSOM can be stored at 2–8 °C for at least 6 months without significantly compromising quality. This study suggests that it is possible to reduce the media inventory by using only mKSOM-R to culture both mouse and rat embryos, and quality media with extended shelf life can be made through modifications.

Lactoferrin affects in vitro and in vivo fertilization and implantationin rats.

Lactoferrin (LF) is present in the oviduct, reduces in vitro gamete interaction, and affects sperm capacitation parameters in humans. Our aim was to investigate LF actions on further stages of the reproductive process in the Wistar rat model. Motile sperm were obtained from cauda epididymis to assess LF binding by direct immunofluorescence and LF effect on acrosome reaction (AR) using a Coomassie blue staining. After ovarian hyperstimulation of female rats, oocytes were surgically recovered and coincubated with motile sperm and different doses of LF to estimate the in vitro fertilization (IVF) rate. To evaluate the LF effect on pregnancy and embryo implantation, female rats (80days old) were placed with males and received daily intraperitoneal injections of LF during one complete estrous cycle (pregnancy experiments) or during the first 8 gestational days (implantation experiments). The number of pregnant females and live born pups was recorded after labor. Moreover, the number of implantation sites was registered during the implantation period. LF was able to bind to the sperm head, midpiece, and tail. 10 and 100μg/ml LF stimulated the AR but reduced the IVF rate. The administration of 100 and 200mg/kg LF significantly decreased the number of implantation sites and the litter size, whereas 100mg/kg LF declined the pregnancy rate. The results suggest that LF might interfere with the reproductive process, possibly interfering with gamete interaction or inducing a premature AR; nevertheless, the mechanisms involved are yet to be elucidated.

Optimized protocols for sperm cryopreservation and in vitro fertilization in the rat.

Laboratory rats have been used in biomedical research for over 170 years. Recently, genome editing technology has facilitated the generation of genetically modified rats worldwide. This development has increased the demand for efficient preservation and production of rat resources. Sperm cryopreservation is the most efficient and robust means to archive genetic resources, and this technique reduces the number of animals required for colony management. Previously, we have reported a protocol for rat sperm cryopreservation and in vitro fertilization using frozen-thawed sperm. Here we describe an improved in vitro fertilization protocol to enhance the fertilization rate of cryopreserved sperm in major strains of rats. In this optimized protocol, treatment of frozen-thawed rat sperm with a high concentration of bovine serum albumin (40 mg/ml) results in a high in vitro fertilization rate. This protocol consists of three main steps: preparation of cryopreserved sperm, in vitro fertilization using cryopreserved sperm and embryo transfer. This process takes approximately 1 month to produce live pups from cryopreserved sperm. This protocol can be easily implemented by researchers and technicians with experience in reproductive engineering technology; it can also be used, albeit with some practice, by researchers and technicians who have no experience in reproductive techniques. This sperm cryopreservation and in vitro fertilization protocol for rats will provide an efficient system for the archiving and production of genetically modified rats for the transgenic community.