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Posts Tagged ‘IVF’


Reporter and Curator: Dr. Sudipta Saha, Ph.D.

 

MicroRNAs (miRNAs) are a group of small non-coding RNA molecules that play a major role in posttranscriptional regulation of gene expression and are expressed in an organ-specific manner. One miRNA can potentially regulate the expression of several genes, depending on cell type and differentiation stage. They control every cellular process and their altered regulation is involved in human diseases. miRNAs are differentially expressed in the male and female gonads and have an organ-specific reproductive function. Exerting their affect through germ cells and gonadal somatic cells, miRNAs regulate key proteins necessary for gonad development. The role of miRNAs in the testes is only starting to emerge though they have been shown to be required for adequate spermatogenesis. In the ovary, miRNAs play a fundamental role in follicles’ assembly, growth, differentiation, and ovulation.

 

Deciphering the underlying causes of idiopathic male infertility is one of the main challenges in reproductive medicine. This is especially relevant in infertile patients displaying normal seminal parameters and no urogenital or genetic abnormalities. In these cases, the search for additional sperm biomarkers is of high interest. This study was aimed to determine the implications of the sperm miRNA expression profiles in the reproductive capacity of normozoospermic infertile individuals. The expression levels of 736 miRNAs were evaluated in spermatozoa from normozoospermic infertile males and normozoospermic fertile males analyzed under the same conditions. 57 miRNAs were differentially expressed between populations; 20 of them was regulated by a host gene promoter that in three cases comprised genes involved in fertility. The predicted targets of the differentially expressed miRNAs unveiled a significant enrichment of biological processes related to embryonic morphogenesis and chromatin modification. Normozoospermic infertile individuals exhibit a specific sperm miRNA expression profile clearly differentiated from normozoospermic fertile individuals. This miRNA cargo has potential implications in the individuals’ reproductive competence.

 

Circulating or “extracellular” miRNAs detected in biological fluids, could be used as potential diagnostic and prognostic biomarkers of several disease, such as cancer, gynecological and pregnancy disorders. However, their contributions in female infertility and in vitro fertilization (IVF) remain unknown. Polycystic ovary syndrome (PCOS) is a frequent endocrine disorder in women. PCOS is associated with altered features of androgen metabolism, increased insulin resistance and impaired fertility. Furthermore, PCOS, being a syndrome diagnosis, is heterogeneous and characterized by polycystic ovaries, chronic anovulation and evidence of hyperandrogenism, as well as being associated with chronic low-grade inflammation and an increased life time risk of type 2 diabetes. Altered miRNA levels have been associated with diabetes, insulin resistance, inflammation and various cancers. Studies have shown that circulating miRNAs are present in whole blood, serum, plasma and the follicular fluid of PCOS patients and that these might serve as potential biomarkers and a new approach for the diagnosis of PCOS. Presence of miRNA in mammalian follicular fluid has been demonstrated to be enclosed within microvesicles and exosomes or they can also be associated to protein complexes. The presence of microvesicles and exosomes carrying microRNAs in follicular fluid could represent an alternative mechanism of autocrine and paracrine communication inside the ovarian follicle. The investigation of the expression profiles of five circulating miRNAs (let-7b, miR-29a, miR-30a, miR-140 and miR-320a) in human follicular fluid from women with normal ovarian reserve and with polycystic ovary syndrome (PCOS) and their ability to predict IVF outcomes showed that these miRNAs could provide new helpful biomarkers to facilitate personalized medical care for oocyte quality in ART (Assisted Reproductive Treatment) and during IVF (In Vitro Fertilization).

 

References:

 

http://link.springer.com/chapter/10.1007%2F978-3-319-31973-5_12

 

http://onlinelibrary.wiley.com/doi/10.1111/andr.12276/abstract;jsessionid=F805A89DCC94BDBD42D6D60C40AD4AB0.f03t03

 

http://www.sciencedirect.com/science/article/pii/S0009279716302241

 

http://link.springer.com/article/10.1007%2Fs10815-016-0657-9

 

http://www.nature.com/articles/srep24976

 

 

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Reporter and Curator: Dr. Sudipta Saha, Ph.D.

 

Mitochondrial disease

 

Mitochondria are present in almost all human cells, and vary in number from a few tens to many thousands. They generate the majority of a cell’s energy supply which powers every part of our body. Mitochondria have their own separate DNA, which carries just a few genes. All of these genes are involved in energy production but determine no other characteristics. And so, any faults in these genes lead only to problems in energy production. Around 1 in 6500 children is thought to be born with a serious mitochondrial disorder due to faults in mitochondrial DNA.

 

Unlike nuclear genes, mitochondrial DNA is inherited only from our mothers. Mothers can carry abnormal mitochondria and be at risk of passing on serious disease to their children, even if they themselves show only mild or no symptoms. It is for such women who by chance have a high proportion of faulty mitochondrial DNA in their eggs for which the methods of mitochondrial replacement or “donation” have been developed. This technique is also referred as the three parent technique and it involves a couple and a donor.

 

Mitochondrial Donation

 

The most developed techniques, maternal spindle transfer (MST) and pro-nuclear transfer (PNT), are based on an IVF cycle but have additional steps. Other techniques are being developed.

 

In both MST and PNT, nuclear DNA is moved from a patient’s egg or embryo containing unhealthy mitochondria to a donor’s egg or embryo containing healthy mitochondria, from which the donor’s nuclear DNA has been removed.

 

mst

Maternal spindle transfer Bredenoord, A and P. Braude (2010) “Ethics of mitochondrial gene replacement: from bench to bedside” BMJ 341.

 

pnt

Pronuclear transfer Bredenoord, A and P. Braude (2010) “Ethics of mitochondrial gene replacement: from bench to bedside” BMJ 341.

 

Research Carried Out and Safety Issues

 

There have been many experiments conducted using MST and PNT in animals. PNT has been carried out since the mid-1980s in mice. MST has been carried out in a wide range of animals. More recently mice, monkeys and human embryos have been created with the specific aim of developing MST and PNT for avoiding mitochondrial disease.

 

  • There is no evidence to show that mitochondrial donation is unsafe
  • Research is progressing well and the recommended further experiments are expected to confirm this view.

 

The main area of research needed is to observe cells derived from embryos created by MST and PNT, to see how mitochondria behave.

 

Concerns about Mitochondrial Donation

 

The scientific evidence raises some potential concerns about mitochondrial donation. Just as we all have different blood groups, we also have different types of mitochondria, called haplotypes. Some scientists have suggested that if the patient and the mitochondria donor have different mitochondrial haplotypes, there is a theoretical risk that the donor’s mitochondria won’t be able to ‘talk’ properly to the patient’s nuclear DNA, which could cause problems in the embryo and resulting child. So, mitochondria haplotype matching in the process of selecting donors may be done to avoid problems.

 

Another potential concern is that a small amount of unhealthy mitochondrial DNA may be transferred into the donor’s egg along with the mother’s nuclear DNA. Studies carried out on MST and PNT show that some so-called mitochondrial ‘carry-over’ occurs. However, the carry-over is lower than 2% of the mitochondria in the resulting embryo, an amount which is very unlikely to be problematic for the children born.

 

References:

 

http://mitochondria.hfea.gov.uk/mitochondria/what-is-mitochondrial-disease/

 

http://mitochondria.hfea.gov.uk/mitochondria/what-is-mitochondrial-disease/new-techniques-to-prevent-mitochondrial-disease/

 

https://www.newscientist.com/article/2107219-exclusive-worlds-first-baby-born-with-new-3-parent-technique/

 

https://www.newscientist.com/article/2108549-exclusive-3-parent-baby-method-already-used-for-infertility/

 

http://www.frontlinegenomics.com/news/7889/ethical-concerns-raised-first-three-parent-ivf-baby/

 

http://www.hfea.gov.uk/docs/2011-04-18_Mitochondria_review_-_final_report.PDF

 

http://www.hfea.gov.uk/docs/Mito-Annex_VIII-science_review_update.pdf

 

http://www.hfea.gov.uk/docs/Third_Mitochondrial_replacement_scientific_review.pdf

 

https://pharmaceuticalintelligence.com/2014/02/26/three-parent-baby-making-practice-of-modifying-oocytes-for-use-in-in-vitro-fertilization-fda-hearing/

 

 

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Reporter and Curator: Dr. Sudipta Saha, Ph.D.

Use of sexed semen in conjunction with in vitro embryo production is a potentially efficient means of obtaining offspring of predetermined sex. Sperm sorting is a means of choosing what type of sperm cell is to fertilize the egg cell. It can be used to sort out sperm that are most healthy, as well as determination of more specific traits, such as sex selection in which spermatozoa are separated into X- (female) and Y- (male) chromosome bearing populations based on their difference in DNA content. The resultant ‘sex-sorted’ spermatozoa are then able to be used in conjunction with other assisted reproductive technologies such as artificial insemination or in-vitro fertilization (IVF) to produce offspring of the desired sex. DNA damage in sperm cells may be detected by using Raman spectroscopy.  It is not specific enough to detect individual traits, however. The sperm cells having least DNA damage may subsequently be injected into the egg cell by intracytoplasmic sperm injection (ICSI).

Sperm sorting utilizes the technique of flow cytometry to analyze and ‘sort’ spermatozoa. During the early to mid 1980s, Dr. Glenn Spaulding was the first to sort viable whole human and animal spermatozoa using a flow cytometer, and utilized the sorted motile rabbit sperm for artificial insemination. Subsequently, the first patent application disclosing the method to sort “two viable subpopulations enriched for x- or y- sperm” was filed in April 1987 and the patent included the discovery of haploid expression (sex-associated membrane proteins, or SAM proteins) and the development of monoclonal antibodies to those proteins. Additional applications and methods were added, including antibodies, from 1987 through 1997. At the time of the patent filing, both Lawrence Livermore National Laboratories and the USDA were only sorting fixed sperm nuclei, after the patent filing a new technique was utilized by the USDA where “sperm were briefly sonicated to remove tails”. USDA in conjunction with Lawrence Livermore National Laboratories, ‘Beltsfield Sperm Sexing Technology’ relies on the DNA difference between the X- and Y- chromosomes.

Prior to flow cytometric sorting, semen is labeled with a fluorescent dye called Hoechst 33342 which binds to the DNA of each spermatozoon. As the X chromosome is larger (i.e. has more DNA) than the Y chromosome, the “female” (X-chromosome bearing) spermatozoa will absorb a greater amount of dye than its male (Y-chromosome bearing) counterpart. As a consequence, when exposed to UV light during flow cytometry, X spermatozoa fluoresce brighter than Y- spermatozoa. As the spermatozoa pass through the flow cytometer in single file, each spermatozoon is encased by a single droplet of fluid and assigned an electric charge corresponding to its chromosome status (e.g. X-positive charge, Y-negative charge). The stream of X- and Y- droplets is then separated by means of electrostatic deflection and collected into separate collection tubes for subsequent processing.

While highly accurate, sperm sorting by flow cytometry will not produce two completely separate populations. That is to say, there will always be some “male” sperm among the “female” sperm and vice versa. The exact percentage purity of each population is dependent on the species being sorted and the ‘gates’ which the operator places around the total population visible to the machine. In general, the larger the DNA difference between the X and Y chromosome of a species, the easier it is to produce a highly pure population. In sheep and cattle, purities for each sex will usually remain above 90% depending on ‘gating’, while for humans these may be reduced to 90% and 70% for “female” and “male” spermatozoa, respectively. Some approaches to in vitro fertilization involve mixing sperm and egg in a test tube and letting nature take its course. But in about half of all infertility cases, a problem with the man’s sperm may require a more direct method. In these cases, a different process, called intracytoplasmic sperm injection (ICSI), in which a single sperm cell is injected directly into an egg, is sometimes used. With this one-shot opportunity, it’s important to choose a sperm cell with the best potential for success. A team at the University of Edinburgh, Scotland, has now announced a new technique to ensure that the best sperm win: analyzing their DNA for potential damage beforehand, and choosing those that are structurally sound.

To optimize success rates of IVF, selection of the most viable embryo(s) for transfer has always been essential, as embryos that are cryopreserved are thought to have a reduced chance of implanting after thawing. Recent developments challenge this concept. Evidence is accumulating that all embryos can now be cryopreserved and transferred in subsequent cycles without impairing pregnancy rates or maybe even with an improvement in pregnancy rates. In such a scenario no selection method will ever lead to improved live birth rates, as, by definition, the live birth rate per stimulated IVF cycle can never be improved when all embryos are serially transferred. In fact, selection could then only lower the live birth rate after IVF. The only parameter that could possibly be improved by embryo selection would be time to pregnancy, if embryos with the highest implantation potential are transferred first.

In the majority of human IVF cycles multiple embryos are created after ovarian hyperstimulation. The viability of these embryos, and as a consequence the chance for an embryo to successfully implant, is subject to biological variation. To achieve the best possible live birth rates after IVF while minimizing the risk for multiple pregnancy, one or two embryos that are considered to have the best chance of implanting are selected for transfer. Subsequently, supernumerary embryos with a good chance of implanting are selected for cryopreservation and possible transfer in the future while remaining embryos are discarded.

The best available method for embryo selection is morphological evaluation. On the basis of multiple morphological characteristics at one or several stages of preimplantation development, embryos are selected for transfer. However, with embryo selection based on morphological evaluation implantation rates in general do not exceed 35%, although varying results have been reported. This has resulted in a strong drive for finding alternative selection methods.

The best studied alternative selection method is preimplantation genetic screening (PGS). The classical form of PGS involves the biopsy at Day 3 of embryo development of a single cell of each of the embryos available in an IVF cycle and analysis of this cell by fluorescence in-situhybridization (FISH) for aneuploidies, for a limited number of chromosomes. Only embryos for which the analyzed blastomere is euploid for the chromosomes tested are transferred. Although this method of PGS has been increasingly used in the last decade, recent trials show that it actually decreases ongoing pregnancy rates compared with standard IVF with morphological selection of embryos.

In an effort to overcome some of the drawbacks of PGS using cleavage stage biopsy and FISH, new methods to determine the ploidy status of a single cell are developed, such as comparative genomic hybridization arrays or single nucleotide polymorphism arrays. Furthermore, in an attempt to avoid the confounding effects of chromosomal mosaicism, embryos are now biopsied at either the zygote or blastocyst stage. In addition, increasing time and money are invested in the development of high-tech, non-invasive methods to select the best embryo for transfer in IVF.

This Include metabolomic profiling, amino acid profiling, respiration-rate measurement and birefringence imaging.

  • In metabolomic profiling, spectrophotometric tests are used to measure metabolomic changes in the culture medium of embryos;
  • in proteomic profiling, proteins produced by the embryo and released into the culture medium are identified;
  • in amino acid profiling, amino acid depletion and production by the embryo is assessed using the culture medium;
  • in respiration-rate measurement, the respiration rate of embryos is assessed; and
  • in birefringence imaging, polarization light microscopy is used to assess the meiotic spindle or the zona pellucida.

Embryo donation (also known as embryo adoption) is the compassionate gifting of residual cryopreserved embryos by consenting parents to infertile recipients. At present, only a limited number of such transactions occur. In 2010, the last year for which U.S. data were available, fewer than 1000 embryo donations were recorded. These acts of giving, unencumbered by federal law, are being guided by a limited number of state laws. Moreover, the practice is sanctioned by professional societies, such as the American Society for Reproductive Medicine, subject to the provision that “the selling of embryos per se is ethically unacceptable.” As such, the not-for-profit donation of existing embryos by consenting parents comports with a triad of commonly held ethical attributes. First, donated embryos are not sold for profit. Second, donated embryos are (by original intent) generated for self-use. Third, donated embryos are the product of an unambiguous parental unit and as such are transferable. All told, embryo donation constitutes an established if limited component of present-day assisted reproduction.

Source References:

http://en.wikipedia.org/wiki/Sperm_sorting

http://www.technologyreview.com/news/411706/best-sperm-for-the-job/

http://humrep.oxfordjournals.org/content/26/5/964.long

http://www.nejm.org/doi/full/10.1056/NEJMsb1215894?query=genetics

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Reporter and Curator: Dr. Sudipta Saha, Ph.D.

 

With the progress in IVF, the contribution of the Fallopian tube towards successful reproduction has been comparatively overlooked. It is clear from the success of IVF, which of course bypasses tubal transport that exposure to the tubal milieu is not an absolute requisite for fertilization or implantation to occur. Thus, the Fallopian tube is often now thought of as little more than a mere conduit. However, in fertilization in vivo, the Fallopian tube plays an essential role in gamete transport, fertilization and the early development of the embryo. It is becoming increasingly evident that the mechanism of tubal transport is much more complex than first thought and can be affected by a wide range of factors and conditions that may impair fertility. The Fallopian tube plays an essential role in tubal transport of both gametes and embryos and in early embryogenesis. The tube undergoes cyclical changes in morphology and ciliary activity in response to ovarian hormones. Whilst the varying contributions to tubal transport of ciliary activity, muscle contractions and secretory activity remain undetermined, there is emerging evidence that muscle contractions may play a role in mixing of secretions rather than in propulsion of gametes and embryos. Ciliary activity is more vigorous in the secretory phase of the menstrual cycle. Many pathological conditions associated with infertility and ectopic pregnancy have been shown either to destroy cilia or to reduce ciliary motion or both. Gonococcal infection produces both destruction of the ciliated cells and reduced ciliary activity, whereas chlamydia also destroys the tubal mucosa. Although the micro-organism itself does not appear to alter ciliary beat, the inflammation and oedema associated with chlamydial salpingitis has been shown to reduce CBF (ciliary beat frequency). Peritoneal fluid from women with mild-and-moderate endometriosis reduces CBF significantly in vitro. An ‘ovum capture inhibitor’ has been described in the peritoneal fluid of women with endometriosis, which covers the fimbrial cilia resulting in a complete but reversible loss of ovum capture ability. The ‘immotile cilia syndrome’ is known to be associated with subfertility. Deciliation is found in Fallopian tubes of women with a past history of ectopic gestation. These women are at increased risk of future tubal pregnancies. This evidence suggests an important role for the tubal cilia in the mechanism of gamete and embryo transport. Further research needs to be undertaken to investigate the functioning of the cilia in vivo. Only one study has measured physiological CBF in vivo, and this needs to be extended to the effect of pathological states on CBF. Direct examination of the effect of conditions such as endometriosis or pelvic inflammatory disease on ovum transport may be possible in animal models using laparoscopy to investigate ovum pick-up and falloposcopy to study ovum transit along the tube. It is only as we begin to understand more about the complex interactions of the effectors of tubal transport that we approach the possibility of being able to improve tubal transport in women afflicted with tubal infertility.

 

Source References:

 

http://humupd.oxfordjournals.org/content/12/4/363.long

 

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