Journal of Reproductive Biotechnology and Fertility

Terato-, astheno- and necrozoospermia negatively influence fertility prognosis in spontaneous conditions or with the use of various assisted reproductive techniques including conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). The correct identification of sperm pathologies will indicate different fertility potentials and outcomes in assisted reproduction technology. Anomalies of only the spermatozoa flagella bear a promising prognosis, but those affecting the sperm chromatin and the neck region entail an increasing chance of failure, which highlights the differential roles played by specific sperm components in fertilization, implantation and early embryonic development. Sperm pathology therefore allows an understanding of abnormal function that goes beyond that provided by classical sperm morphology classifications that are mainly based on descriptions of abnormal sperm shapes with no insight into the mechanisms or the pathological details.

This review article gives a brief history of the classical experiments that led to the development of the embryo culture medium and in vitro embryo culture. It proposes that, in view of the outstanding and significant pioneering contributions of Wesley Kingston Whitten to the development of embryo culture medium, he be considered the “Father of Embryo Culture Medium” . Furthermore, it describes the nutritional requirements of early embryos and how these requirements with specific references to carbohydrates, amino acids, phosphates, growth factors, etc, have been utilized to formulate increasingly more complex embryo culture media. This has led to the development of progressively more efficacious embryo culture media including the formulation of completely defined and synthetic protein-free embryo culture medium. The review also describes physical factors, growth factors, insemination methods for the fertilization of oocytes and culture methods affecting embryo growth, development, metabolism, oxygen embryotoxicity and survival. In procedural terms, the review also summarizes the evolution of embryo culture techniques from tube culture to, microdrop culture under oil to co-culture to ultra microdrop culture techniques. It includes techniques of in vitro maturation and for the selection of potentially viable embryos of various developmental stages.

Oocytes collected from patients following ovarian stimulation exhibit varying qualities. The quality of oocytes has an effect on its subsequent fertilization, developmental competence post-fertilization and the viability of resultant embryos. The aim of this article was to review the morphological criteria devised for assessment of oocyte quality by ordinary light and polarized light microscopy before IVF or ICSI. The parameters employed in the evaluation of oocyte morphology include the appearance of: structure of the cumulus–oocyte complex, oocyte cytoplasm, perivitelline space, zona pellucida, polar body and meiotic spindle.

Since Intra-uterine insemination IUI requires the isolation of motile spermatozoaatozoa, advances in andrology research has helped understand the physiology of male germ cell and allowed development of better and more sophisticated techniques to separate functional spermatozoaatozoa from those that are immotile, have poor morphology or are not capable to fertilize oocytes. When compared with other techniques, Density Gradients Centrifugation (DGC) technique allows maximum yield of motile spermatozoa. Several density media like IxaPrep, Nycodenz, SilSelect, PureSpermatozoa and Isolate have been developed to replace Percoll which, was banned in 1996 due to risk of contamination with endotoxins. Semen analysis, according to the revised, WHO (2010) criteria should be carried out prior to processing the sample. Although sophisticated testing—such as DNA fragmentation analysis, oxidative stress analysis and spermatozoa evaluation for genomic, proteomic and metabolic factor are in research phase, it is more than likely that in future these will help in assessing the suitability of the sample in certain cases of male factor or unexplained infertility. The isolation of functional spermatozoaatozoa from highly viscous ejaculates is a challenge that can be performed enzymatically to liquefy the ejaculate. Special care should be taken when processing HIV, Hep B and Hep C, positive samples. Prior to insemination, the processed sample should be tested to ensure the absence of HIV, Hep B and Hep C. There is no agreement between Andrologists as to what should be the minimum motile count for IUI to be successful. Pregnancies have been reported with counts in range of 1 – 10 million motile spermatozoas. Morphology of the processed sample has limited influence on the final outcome. Finally, single insemination 40 h after the hCG injection should be performed using a soft catheter. Normally, 2nd insemination 24 hrs later is only indicated when the follicle has not ruptured at 40 h post hCG injection.

After more than 30 years following the birth of Louise Brown, the field of assisted reproduction techniques (ART) is still burdened by low pregnancy rates and relatively high multiple pregnancy rates. Embryo selection is still based on morphological criteria and any new technologies that would increase the ability to identify the most viable gametes and embryos would certainly improve ART outcome. Metabolomic profiling of both gametes and embryos appears to be highly promising to achieve this goal. The review will focus on the several applications of metabolomics in ART, where this quantitative, non-invasive, rapid approach could result in the generation of metabolite markers with high clinical impact. Nevertheless, extensive validation studies are needed before embryo screening tests based on metabolomic profiling become available for clinical use.

Vitrification is a cryopreservation technique increasingly applied in clinical practice for cells and tissue. This review article focuses mainly on the efficiency of vitrification of human reproductive cells and tissue, by analysing the clinical results reported in the literature. The second aspect discussed is safety of vitrification procedure. Different procedures and different types of carriers can be used, and in some cases vitrification requires a direct contact between cell/tissue/carrier and liquid nitrogen; this causes concern regarding the safety of this cryopreservation technique. Although the risk of contamination during cryopreservation remains negligible, this article explains how to overcome the hypothetical risk of contamination when using different types of vitrification carriers, in order to satisfy all existing directives.

The co-culture technique was first implemented in animal models in the mid-sixties in an attempt to improve the suboptimal conditions provided by culture media. Since then, autologous endometrial co-culture was introduced in human IVF, and different outcomes were reported regarding blastulation, fragmentation and implantation rates. Various growth factors and cytokines were proposed to be involved in the mechanism by which co-culture systems improved embryo development. However, randomized controlled trials are still warranted to provide evidence on the efficacy of endometrial co-cultures in the era of modern sequential- and monoculture media.

In the United States, the Clinical Laboratory Improvement Act (CLIA) of 1988 describes requirements and guidelines for implementing a quality control/quality assurance (QC/QA) program for moderate and high complexity laboratories. These requirements and guidelines apply to Assisted Reproductive Technology (ART) laboratories as well. The general topic of QC and QA as it pertains to in vitro fertilization (IVF) and embryo transfer (ET) is extensively reviewed. This review summarizes many of the QC and QA events that contribute to the advancement of knowledge in this biotechnological field. These events include control of the culture environment inside and outside of the incubator, as well as factors that affect culture media. This review also discusses, in considerable detail, the QC and the QA that pertain to equipment used within the laboratory and how to control for potential contaminants, which reside within the laboratory. This review provides evidence to indicate the need for laboratory personnel to monitor quality improvement issues on a continuous basis. Personnel must be willing to change as improvements in technology occur in order to meet the ever-evolving demands of a more difficult patient population. Suggestions for meeting these demands are offered.

The College of American Pathologists (CAP) serves over 300 reproductive laboratories in four countries. The goal of CAP is to improve laboratory conditions for the patient and the personnel who work in the laboratory. CAP accomplishes this goal through proficiency testing and inspections. Qualified inspectors will arrive, at a prearranged time, to inspect a laboratory. They will review procedure manuals, inspect reagents, instruments and equipment, and verify calibration of instruments/equipment. In addition, they will inspect the laboratory to insure the safety of the patients, their gametes, and the safety of the laboratory personnel. A summary report will be generated by the inspectors as to their findings with written reports given to the laboratory director and to CAP. It is through such organizations as CAP that the quality of patient results and the safety and well-being of the patient and the laboratory personnel have been improved over the years.

In oviparous animals, the egg hatches outside of the body and is exposed to light; in some cases throughout the development of the fetus. In mammals, fertilization and the growth of embryos in vivo occurs in the dark but in human IVF, these embryos are exposed to variable light sources and intensities. Light can affect embryonic development in some species via either a direct toxic effect on the embryo, or indirectly via photo-oxidation of components in the media or oil. Although data regarding the effect of light on human embryos is lacking, it is prudent to take appropriate steps to minimize the potential harmful effects of both ambient and microscopic light on embryos.

The purpose of this investigation was to assess the incidence of oocyte damage (oocyte lysis) following intracytoplasmic sperm injection (ICSI) and its association with oocyte quality and with the personnel performing the procedure. Data concerning damage were accumulated from a laboratory database that tabulated the results of each patient's ICSI results. The incidence of lysis following ICSI was 4.8% of all injected oocytes. The number of oocytes injected and the incidence of intact (not lysed) oocytes that were fertilized by ICSI both were inversely correlated with the incidence of lysis. Individual technicians had distinctively different profiles of lysis, 1PN and 3PN embryos. Technicians in training had slightly elevated incidence of lysis. Whereas evidence is presented that both patient-specific oocyte quality and technician-specific performance are significantly related to the incidence of lysis, the small incremental incidence of lysis associated with each source leads us to believe that the incidence of lysis is largely unpredictable and unsystematic in nature.

The objective of this study was to characterize mongrel dog seminal plasma proteins with SDS-PAGE and to determine the correlation between different proteins and semen characteristics. Ejaculate volume, sperm motility, total sperm count, live sperm percent, percentage abnormalities and membrane integrity (hypo osmotic swelling test) were assessed in 5 ejaculates of seven dogs. For each dog, seminal plasma was also pooled from five ejaculates and proteins were separated by SDS-PAGE using polyacrylamide concentration of 13% and 15% in separating gels. There was considerable variation in the semen characteristics among the ejaculates of different dogs. Total protein content of seminal plasma varied from 12.06 to 21.3 mg/ml in different dogs. The number of protein bands ranged from 10–12 in different dogs. The proteins with mol wt of 42, 33, 29, 24 and 14.0 kDa were major proteins in seminal plasma of mongrel dog and ranged from 61.3% to 74.3% in different dogs. There were differences in live sperm percent (CD 5% =21.7) and HOS positive spermatozoa (CD 5% =21.01) among different dogs. There was strong correlation between volume of semen and total sperm concentration (r=+0.87), whereas the correlation between motility vs HOST (r=+0.58) and motility vs live percent (r=+0.60) was moderate. A positive correlation was observed between concentrations of 82, 70, 24, 14 kDa proteins vs percent motility, live sperm percent and percent HOS positive spermatozoa. Our study confirmed the previous conclusion that HOS-test could also be included in routine evaluation of semen. The positive correlation of 14, 24, 70 and 82 kDa proteins with semen characteristics also inferred the role of these proteins in the fertility of mongrel dog semen, which needs to be worked out further for their role in the process of fertilization.

The aim of this study is to compare blastocyst development and cycle outcomes of good prognosis elective single embryo transfer patients using conventional or time lapse incubators. A randomized study was designed to evaluate embryo development until the blastocyst stage in either conventional or time-lapse incubators in good prognosis patients. Furthermore cycle outcomes of the patients were evaluated. The study involved 64 patients (33 for time-lapse and 31 for conventional incubation) in their first or second treatment cycle, with no recurrent spontaneous miscarriages, age<35 years, body mass index <28 kg/m² and ≥8 oocytes retrieved. The primary outcome was the proportion of good and top quality blastocysts on day 5 and the secondary outcome was the implantation and pregnancy rates after elective single embryo transfer in good prognosis patients. The analysis performed revealed no statistical difference between both groups for blastocyst development, implantation and clinical pregnancy rates. In a selected good prognosis elective single embryo transfer population, similar rates of good and top quality blastocysts and comparable implantation and pregnancy rates were achieved. However, the stable culture conditions provided by time-lapse incubators and the use of morphokinetic parameters may allow the selection of the best available embryos with high developmental capacity, especially in poor prognosis patients.

In the field of nuclear transfer handmade cloning (HMC) technique has been a relatively recent addition. In the present investigation attempts were made to efficiently derive stem cell from hand made cloned (HMC) embryos in goat using adult fibroblast donor cell and to characterize the derived putative ntES cell for their stemness. Oocytes were collected from slaughterhouse ovaries and matured in maturation medium containing TCM199 (HEPES modified), 1 μg/ml FSH, 10 μg/ml LH, 1 μg/ml Estradiol-17β, 3% BSA and 5% FBS. The efficiency of the donor fibroblast cell for nuclear transfer was also assessed and an overall cleavage and morula formation rate of 62.44%±3.9 & 35.30%±3.86 was obtained respectively. Genetic relationship between the cloned embryos and donor cells was established by PCR amplification of highly polymorphic goat major histocompatibility complex (MHC) class II DRB gene. The PCR products were further subjected to SSCP for confirming the genetic relationship conclusively. Stem cell colonies were successfully derived from HMC embryos obtained from fibroblast donor cell. Primary colony formation rate was 61.66%±4.62 for fibroblast donor cell derived embryos. The putative ntES colonies were positively characterized for extracellular markers like alkaline phosphatase, TRA-1-60, TRA-1-81, SSEA-1, SSEA-4 by immunocytochemistry and intracellular markers like Oct4, Sox2 and Nanog with reverse-transcription-PCR. The differentiating ability of the derived cells was tested by embryoid body formation. Then the derived putative ntES cells were successfully cryopreserved for further investigation. The study has established an efficient protocol for putative ntES cell derivation from HMC embryos using adult fibroblast as a donor cell. It could be concluded that the present investigation has substantial significance as patient specific ntES cells have proven their therapeutic significance.

The transport of cryopreserved biological products at a consistent temperature of −196°C can present significant challenge to researchers and laboratory personnel that may have little experience in this area. This communication will explore the basic principles for successfully managing the transport of cryogenic shipments both domestically and internationally, examining the need for proper packaging, knowledge of the regulatory environment in the country of destination, and a sound risk management strategy. It will also examine the special circumstances and considerations associated with sending cryopreserved biological samples to emerging nations and how best to safeguard shipments in the face of frail infrastructure, cumbersome regulatory environments, dramatic temperature fluctuations, etc. Finally, it will provide researchers with guidelines on identifying reliable and experienced transport/logistics providers and ensuring that their service capabilities conform to individual shipping needs.

During infertility treatment with IVF, embryos are cultured either in groups or individually. Each approach has potential benefits and detriments, and the purpose of this review is to try to come to a consensus based on the literature as to which approach yields superior results. Group culture of embryos may produce better quality embryos via secretion of embryotrophic factors, while opponents of the approach argue that embryos cultured together may either deplete the media of substrates or negatively affect nearby embryos via the transmission of other secreted factors. In these cases, quantity of embryos, volume of media and proximity and quality of companion embryos are also important factors to consider. While it has long been accepted that group culture is beneficial for embryos from various animal species, emerging data also suggest a similar benefit in the human. Conversely, embryos cultured individually avoid potential substrate depletion, negative impact from factors secreted from companion embryos, while more practically permitting the ability to monitor and track the embryo for identification via morphology or molecular analysis to select and transfer potentially superior embryos. Importantly, advancements in embryo culture platforms now permit tracking of individual embryos, while also offering ability to reap the benefits of group culture. These approaches utilize confined microenvironments immediately surrounding the embryos that may be conducive for periodic sampling/analysis, while also allowing access to a larger media reservoir to avoid substrate depletion. Thus, though questions remain as to optimal embryo density and volume of culture media, group embryo culture in the correct culture platform is likely to be superior to individual embryo culture.