In the present report we studied the safety, efficacy and efficiency of using an infrared laser to facilitate IVF by assessing fertilization, development and birth rates after laser-zona drilling (LZD) in 30 subfertile genetically modified (GM) mouse lines. We determined that LZD increased the fertilization rate four to ten times that of regular IVF, thus facilitating the derivation of 26 of 30 (86.7%) GM mouse lines. Cryopreserved two-cell stage embryos derived by LZD-assisted IVF were recovered and developed to blastocysts in vitro at the same rate as frozen–thawed embryos derived by regular IVF. Surprisingly after surgical transfer to pseudopregnant recipients the birth rate of embryos derived by LZD-assisted IVF was significantly lower than that of embryos derived by regular IVF. However this result could be completely mitigated by the addition of 0.25 M sucrose to the culture medium during LZD which caused the oocyte to shrink in volume relative to the perivitelline space. By increasing the distance from the laser target site on the zona pellucida, we hypothesize that the hyperosmotic effect of sucrose reduced the potential for laser-induced cytotoxic thermal damage to the underlying oocytes. With appropriate preparation and cautious application, our results indicate that LZD-assisted IVF is a safe, efficacious and efficient assisted reproductive technology for deriving mutant mouse lines with male factor infertility and subfertility caused by sperm–zona penetration defects.
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Ming-Wen Li, Kristy L Kinchen, Jadine M Vallelunga, Diana L Young, Kaleb D K Wright, Lisa N Gorano, Katherine Wasson, and K C Kent Lloyd
R John Aitken
There has never been a greater need for scientists trained in reproductive science. Most developed countries are witnessing unprecedented rates of recourse to assisted conception sitting cheek-by-jowl with high rates of induced abortion. This article addresses these two incongruous faces of reproductive healthcare. Every year at least 44 million abortions are performed worldwide, many under unsafe and insanitary conditions that carry a significant risk to the lives of women deprived of safe, effective methods for controlling their fertility. Although birth control is a complex issue involving myriad social and political factors, the technical vacuum in this area is significant. Through no fault of the family planning authorities, there have been no radically new methods of fertility control since the oral contraceptive pill was introduced in 1960 and even this contribution to planned parenthood has its roots in the biochemistry of the 1920s and 1930s. Moreover, the pharmaceutical industry has, by and large, turned its back on fundamental research activities in this area. At present, our major investment in reproductive healthcare involves treating ever-increasing numbers of couples with assisted reproductive technologies (ART). However, these treatments are often delivered without critically considering the underlying causes of this condition or seriously contemplating the long-term consequences of the current enthusiasm for such therapy. Significantly, the clinical factors underpinning the commitment of couples to ART include advanced maternal age and a variety of lifestyle factors, such as smoking and obesity, which are known to compromise the developmental potential of the oocyte and DNA integrity in spermatozoa.
Kuan-Hao Tsui, Peng-Hui Wang, Li-Te Lin, and Chia-Jung Li
Because ovarian granulosa cells are essential for oocyte maturation and development, we validated human granulosa HO23 cells to evaluate the ability of the DHEA to prevent cell death after starvation. The present study was aimed to investigate whether DHEA could protect against starvation-induced apoptosis and necroptosis in human oocyte granulosa HO23 cells. The starvation was induced by treatment of serum-free (SF) medium for 4 h in vitro. Starvation-induced mitochondrial depolarization, cytochrome c release and caspase-3 activation were largely prevented by DHEA in HO23 cells. We found that treatment with DHEA can restore starvation-induced reactive oxygen species (ROS) generation and mitochondrial membrane potential imbalance. In addition, treatment of DHEA prevents cell death via upregulation of cytochrome c and downregulation of BAX in mitochondria. Most importantly, DHEA is ameliorated to mitochondrial function mediated through the decrease in mitochondrial ROS, maintained mitochondrial morphology, and enhancing the ability of cell proliferation and ROS scavenging. Our present data strongly indicate that DHEA reduces programmed cell death (apoptosis and necroptosis) in granulosa HO23 cells through multiple interactions with the mitochondrion-dependent programmed cell death pathway. Taken together, our data suggest that the presence of DHEA could be beneficial to protect human oocyte granulosa HO23 cells under in vitro culture conditions during various assisted reproductive technology (ART) programs.
Free Chinese abstract: A Chinese translation of this abstract is freely available at http://www.reproduction-online.org/content/154/2/101/suppl/DC1
Sandra Laurentino, Jennifer Borgmann, and Jörg Gromoll
The influence of epigenetic modifications on reproduction and on the function of male germ cells has been thoroughly demonstrated. In particular, aberrant DNA methylation levels in sperm have been associated with abnormal sperm parameters, lower fertilization rates and impaired embryo development. Recent reports have indicated that human sperm might be epigenetically heterogeneous and that abnormal DNA methylation levels found in the sperm of infertile men could be due to the presence of sperm populations with different epigenetic quality. However, the origin and the contribution of different germ cell types to this suspected heterogeneity remain unclear. In this review, we focus on sperm epigenetics at the DNA methylation level and its importance in reproduction. We take into account the latest developments and hypotheses concerning the functional significance of epigenetic heterogeneity coming from the field of stem cell and cancer biology and discuss the potential importance and consequences of sperm epigenetic heterogeneity for reproduction, male (in)fertility and assisted reproductive technologies (ART). Based on the current information, we propose a model in which spermatogonial stem cell variability, either intrinsic or due to external factors (such as endocrine action and environmental stimuli), can lead to epigenetic sperm heterogeneity, sperm epimutations and male infertility. The elucidation of the precise causes for epimutations, the conception of adequate therapeutic options and the development of sperm selection technologies based on epigenetic quality should be regarded as crucial to the improvement of ART outcome in the near future.
K I Aston, C M Peterson, and D T Carrell
Twin birth rates have increased markedly in developed countries since the 1970s for two primary reasons: increasing maternal age and the advent and increasing use of fertility treatments. In addition, monozygotic (MZ) twin pregnancies have been reported to occur at a significantly higher rate following assisted reproductive technologies (ART) procedures compared with the natural incidence. Twin pregnancies are of concern due to a dramatically increased risk of associated complications. Monozygotic twin pregnancies carry a 10–20% risk of twin–twin transfusion syndrome, and monoamniotic monochorionic twins are additionally at risk for cord entanglement. While the mechanisms and contributory factors for dizygotic twinning are well established, very little is known about the mechanisms involved in MZ twinning or the factors that contribute to its occurrence. In this review, we will discuss a number of potential mechanisms involved in MZ twinning and explore factors that may be contributing to the increased incidence of ART-associated MZ twins. An improved understanding of the factors that contribute to increased MZ twinning associated with ART will help to elucidate the poorly understood mechanisms involved in the process and will further aid in reducing the overall incidence of multiple pregnancies with their associated risks following ART procedures.
Georgios Anifandis, Eleni Koutselini, Ioannis Stefanidis, Vassilios Liakopoulos, Constantinos Leivaditis, Themis Mantzavinos, and Nikolaos Vamvakopoulos
This prospective study was undertaken to reassess the prognostic value of leptin during critical stages of in vitro fertilization-embryo transfer (IVF-ET) and address its role in the functional staging of assisted reproductive technologies at the level of embryo quality. Serum and follicular fluid samples of 100 selected women undergoing the long IVF-ET protocol were collected for leptin and embryo quality determination. The highest serum leptin concentration (52.11 ± 4.27 ng/ml) was observed on ovum pick up day, while follicular fluid leptin was higher than all serum samples examined (62.59 ± 5.73 ng/ml). Serum leptin above 59.48 ± 7.6 ng/ml was associated with ‘poor’ embryo quality and above 56.87 ± 5.52 ng/ml with pregnancy failure. Elevated leptin concentrations were associated with reduced ovarian stimulation and response, follicle maturation, embryo quality and pregnancy success. Our findings suggest that leptin modulates embryo quality and may serve as a sensitive marker of IVF outcome.
Angela L Schenewerk, Francisco Í Ramírez, Christopher Foote, Tieming Ji, Luis A Martínez-Lemus, and Rocío Melissa Rivera
Maternal obesity and the use of assisted reproductive technologies (ART) are two suboptimal developmental environments that can lead to offspring obesity and cardiovascular disease. We hypothesized that these environments independently and synergistically adversely affect the offspring's weight and cardiovascular performance at ∼7 weeks of age. Mice were fed either 24% fat and 17.5% high-fructose (HF) corn syrup or maintenance chow (5% fat; low-fat, no-fructose (LF)). Dams were subdivided into no ART and ART groups. ART embryos were cultured in Whitten's medium and transferred into pseudopregnant recipients consuming the same diet as the donor. Offspring were fed the same diet as the mother. Body weights (BW) were measured weekly and mean arterial pressure (MAP) was collected through carotid artery catheterization at killing (55±0.5 days old). Expression of genes involved in cardiovascular remodeling was measured in thoracic aorta using qRT-PCR, and levels of reactive oxygen species (ROS) were measured intracellularly and extracellularly in mesenteric resistance arteries. ART resulted in increased BW at weaning. This effect decreased over time and diet was the predominant determinant of BW by killing. Males had greater MAP than females (P=0.002) and HF consumption was associated with greater MAP regardless of sex (P<0.05). Gene expression was affected by sex (P<0.05) and diet (P<0.1). Lastly, the use of ART resulted in offspring with increased intracellular ROS (P=0.05). In summary, exposure to an obesogenic diet pre- and/or post-natally affects weight, MAP, and gene expression while ART increases oxidative stress in mesenteric resistance arteries of juvenile offspring, no synergistic effects were observed.
Esther W Kabithe and Ned J Place
Fertility and fecundity decline with advancing age in female mammals, but reproductive aging was decelerated in Siberian hamsters (Phodopus sungorus) raised in a short-day (SD) photoperiod. Litter success was significantly improved in older hamsters when reared in SD and the number of primordial follicles was twice that of females held in long days (LD). Because anti-Müllerian hormone (AMH) appears to inhibit the recruitment of primordial follicles in mice, we sought to determine whether the expression patterns of AMH differ in the ovaries and serum of hamsters raised in SD versus LD. Ovaries of SD female hamsters are characterized by a paucity of follicular development beyond the secondary stage and are endowed with an abundance of large eosinophilic cells, which may derive from granulosa cells of oocyte-depleted follicles. In ovaries from 10-week-old SD hamsters, we found that the so-called ‘hypertrophied granulosa cells’ were immunoreactive for AMH, as were granulosa cells within healthy-appearing primary and secondary follicles. Conversely, ovaries from age-matched LD animals lack the highly eosinophilic cells present in SD ovaries. Therefore, AMH staining in LD was limited to primary and secondary follicles that are comparable in number to those found in SD ovaries. The substantially greater AMH expression in SD ovaries probably reflects the abundance of hypertrophied granulosa cells in SD ovaries and their relative absence in LD ovaries. The modulation of ovarian AMH by day length is a strong mechanistic candidate for the preservation of primordial follicles in female hamsters raised in a SD photoperiod.
Jose R Rodriguez-Sosa, Guilherme M J Costa, Rahul Rathi, Luiz R França, and Ina Dobrinski
Testis tissue xenografting is a powerful approach for the study of testis development and spermatogenesis, and for fertility preservation in immature individuals. In bovine testis xenografts, maturation and spermatogenesis are inefficient when compared to other species. To evaluate if exogenous modulation of the endocrine milieu in recipient mice will affect spermatogenic efficiency in xenografts from newborn calves, recipient mice were treated with the GnRH antagonist acyline (5 mg/kg s.c. every 2 weeks) to reduce testosterone production in xenografts, or with 6-N-propyl-2-thiouracil (PTU, 0.1% in drinking water for 4 weeks), to induce transient hypothyroidism in recipient mice respectively. Both treatments altered developmental parameters of testis xenografts and reduced germ cell differentiation. While the effects of acyline treatment can be attributed to inhibition of GnRH and gonadotropin action, lower Sertoli cell numbers and decreased seminiferous tubule length observed after PTU treatment were opposite to effects reported previously in rats. Regardless of treatment, Sertoli cells underwent only partial maturation in xenografts as Müllerian inhibiting substance and androgen receptor expression were lower than in donor and adult tissue controls respectively. In conclusion, although treatments did not result in improvement of maturation of bovine testis xenografts, the current study demonstrates that exogenous modulation of the endocrine milieu to affect xenograft development in recipient mice provides an accessible model to study endocrine control of spermatogenesis in large donor species.
Xavier Druart, Jean-Luc Gatti, Sylvie Huet, Jean-Louis Dacheux, and Patrice Humblot
Hypotonic resistance of boar spermatozoa was investigated by measuring the ratio of live/dead spermatozoa (SYBR-14/propidium iodide) by flow cytometry after hypotonic stress. The survival rate of ejaculated spermatozoa incubated in hypotonic solutions ranging from 3 to 330 mmol/kg followed a sigmoid curve that fitted a simple logistic model. The critical osmolality value (Osmcrit) at which 50% of spermatozoa died was determined with this model. Hypotonic resistance of spermatozoa increased with temperature between 15 and 39 °C and decreased after hydrogen superoxide treatment, but was not modified during 8 days of preservation in Beltsville thawing solution. Hypotonic resistance markedly decreased during epididymal maturation and after ejaculation as Osmcrit at 15 °C was 54.7±3.2, 68.5±10.6, 116.7±2.1 and 194.3±3.7 mmol/kg for the caput, corpus, cauda and ejaculated spermatozoa respectively. Hypo-osmotic stress of 100 mmol/kg revealed a sperm subpopulation exhibiting increased hypotonic resistance compared with the whole ejaculate (Osmcrit=67.8±2.1 mmol/kg). Consistent differences were observed between lean and standard breeds (Pietrain versus Large White) and between boars within the same breed. According to data collected by artificial insemination centers during a large-scale field trial, hypotonic resistance of ejaculates was found to be positively correlated with in vivo fertility.