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In Vitro Fertilization Options
Daemon
Posted: Friday, December 12, 2014 12:00:00 AM
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In Vitro Fertilization

This technique is used for the conception of a human embryo outside the mother's body. Several ova—or eggs—are removed from the mother's body and placed in special laboratory culture dishes. Sperm from the father are then added. If fertilization occurs, the fertilized ovum, after undergoing several cell divisions, is either transferred to the mother's or a surrogate mother's body for normal development in the uterus, or is frozen for later implantation. What does in vitro mean in Latin? More...
L.Rai
Posted: Friday, December 12, 2014 12:11:24 AM

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In Vitro means:

....from the Latin meaning in glass, is used, because early biological experiments involving cultivation of tissues outside the living organism from which they came, were carried out in glass containers such as beakers, test tubes, or petri dishes.

Today, the term in vitro is used to refer to any biological procedure that is performed outside the organism it would normally be occurring in, to distinguish it from an in vivo procedure, where the tissue remains inside the living organism within which it is normally found.
NeuroticHellFem
Posted: Friday, December 12, 2014 5:23:45 AM

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If you haven't read Brave New World by Aldous Huxley, read it!
JUSTIN Excellence
Posted: Friday, December 12, 2014 5:48:58 AM

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Since the world's first "test-tube" baby was born in Great Britain in 1978, we have witnessed a tremendous progress in the field of human ARTs [Assisted Reproductive Technologies] which is reflected in the high rates of success accomplished in infertility treatments. Despite these advances, live birth rates achieved by assisted conception remain relatively low and could be improved. In light of the known influence of the fertilizing spermatozoon not only on early but also on late embryonic development, selection of the best sperm from heterogeneous sperm samples would impact positively on the outcomes of human ARTs. Accurate identification of normal/healthy spermatozoa is of especial importance during ICSI (Intracytoplasmic Sperm Injection), in which a sperm cell is deliberately injected into the mature oocyte by the technician bypassing all natural barriers. There is great concern about the risk of using sperm with chromosomal abnormalities and/or damaged DNA what can lead to inadvertently transmission of genetic diseases to the offspring. Therefore, improvements of the available sperm selection techniques and/or development of new methods for precise sperm selection are highly desirable. Despite encouraging preliminary results obtained with advanced sperm selection techniques, more research is warranted to address safety issues before widespread application of these methods. In this regard, animal models can provide answers to important safety concerns related to the introduction of advanced and emerging methods for sperm selection into human ART.



Quote:
Detailed theoretical analysis, using mathematical modeling, reveals that sex is a very inefficient way of reproducing. The inefficiency lies in making male offspring. In the majority of sexual species, only the female contributes energy and resources to the young. In contrast the males rarely contribute more than the minimum - a tiny sperm carrying genes, but devoid of other resources.
— The Evolution of Life, edited by Linda Gamlin and Gail Vanes, Oxford University Press, New York, 1987


Major progress has been achieved in spermatology in the last decade, including the successful transplantation of fresh and cryopreserved testicular tissues, reported production of spermatogenic cells from stem cells, production of transgenic animals and routine use of intracytoplasmic sperm injection (ICSI) in clinical practice. In this post, we attempt to review some of the advances in the sperm capacitation field and ARTs in the last years.

Preimplantation development in mammalian species is a challenging stage of embryogenesis, which makes in vitro fertilization and embryo culture sophisticated biotechniques. One of the main tasks is to provide cultured embryos an adequate environment that allows embryonic-maternal transition, and consequent achievement of an independent gene expression program. In this respect, mammalian models have been used to further elucidate the mechanisms of embryonic genome activation. Within the main players in this context we can include chromatin compactor/relaxing agents, and among them histone modifying enzymes and their consequent posttranslational modifications.



In Vitro Production (IVP) of bovine embryos

Development of bovine embryos in vitro for research purposes is mainly carried out using oocytes obtained from slaughterhouse ovaries. Blastocyst production includes oocyte maturation, in vitro fertilization and embryo culture, and can be accomplished in 7-8 days, with rates of approximately 40%. Here we briefly describe all steps of this process.

Supplements

Reagents and culture media were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise stated.

Preparation and selection of oocytes

Bovine ovaries were collected at a local slaughterhouse and processed within 2 h after slaughter. The ovaries were washed in saline (37ºC) and follicles measuring 3 to 8 mm in diameter were aspirated with an 18-gauge needle coupled to a 20-mL syringe. Follicle liquid was placed in a 50 mL conic tube for 20 min sedimentation at 37ºC, and then 10 mL of the sediment was collected and transferred to 100 mm Petri dishes. Cumulus-oocyte complexes (COCs) presenting at least three layers of cumulus cells and homogenous cytoplasm were selected under a stereomicroscope. The COCs were washed in HEPES-buffered TCM-199 (Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal calf serum (FCS; Cripion Biotecnologia, Andradina, SP, Brazil), 16 μg/mL sodium pyruvate and 83.4 μg/mL amikacin (Instituto Biochimico, Rio de Janeiro, RJ, Brazil).

In vitro maturation (IVM)

Groups of 15 COCs were transferred to 100-μL drops of medium containing sodium bicarbonate-buffered TCM-199 supplemented with 10% FCS, 1.0 μg/mL FSH (Folltropin™, Bioniche Animal Health, Belleville, ON, Canada), 50 μg/mL hCG (Profasi™, Serono, Sao Paulo, SP, Brazil), 1.0 μg/mL estradiol, 16 μg/mL sodium pyruvate and 83.4 μg/mL amikacin, covered with sterile mineral oil (Dow Corning Co., Midland, MI, USA) and incubated for 24 h at 38.5°C in an atmosphere of 5% CO2 in air under saturated humidity.

In vitro fertilization (IVF)

After in vitro maturation (IVM) the cumulus cells were partially removed from the oocytes by vigorous pipetting. Groups of 20 oocytes were washed twice and transferred to 80-μL drops of TALP-IVF medium supplemented with 0.6% BSA, 10 μg/mL heparin, 18 μM penicillamine, 10 μM hypotaurine and 1.8 μM epinephrine, and covered with sterile mineral oil. A frozen straw of semen was thawed at 35.5°C and centrifuged on a discontinuous 45/90 Percoll gradient for 7 min at 3600 x g. The pellet was collected (100 μL) and resuspended in 700 μL TALP-IVF medium and again centrifuged for 5 min at 520 x g. After centrifugation, 30 μL of the medium containing the pellet was collected from the bottom of the tube and homogenized in a conical tube. The suspension was adjusted for a final concentration of approximately 104 mobile spermatozoa for each oocyte. The plates were incubated at 38.5°C for 20 h in an atmosphere of 5% CO2 in air under saturated humidity. Semen from the same bull and the same batch was used for all replicates.

In vitro culture (IVC)

After IVF, presumptive zygotes were denuded of cumulus cells by vigorous pipetting. Embryos were washed three times and transferred in groups of 15 to 20 to be cultured in 100-μL drops of SOF medium supplemented with 5 mg/mL BSA and 2.5% FCS. The dishes were then incubated in an atmosphere of 5% O2 in air under saturated humidity for 5 days at 38o C. The cleavage rate, blastocyst development, and blastocyst hatching were evaluated 48 h, 7 days and 9 days after IVF, respectively.

Immunocytochemistry of H3K9ac and H3k27me3

For this experiment, we used day 5 embryos, 70h after IVF. At this timepoint, approximately 30-50% of embryos should be at the 5th cell cycle. Embryos were fixed in 4% paraformaldehyde for 30 min at 37°C and stored at 4°C in PBS supplemented with 3% BSA and 0.5% Triton X-100 for up to 1 week. Fixed embryos were incubated in blocking solution (3% BSA and 0.2% Tween-20 in PBS) for 1 h at room temperature. Next, the embryos were incubated with the primary antibodies (mouse anti-H3K9ac monoclonal antibody, 1:200, and rabbit anti-H3k27me3 monoclonal antibody (1:200; Upstate Biotechnology, Lake Placid, NY, USA) for 12h at 4°C. The embryos were then washed three times in PBS for 10 min and incubated with the secondary antibody (chicken anti-mouse-alexa 488; 1:200; Invitrogen Molecular Probes, Eugene, OR, EUA), and goat anti-rabbit-alexa 555 (1:200; Invitrogen Molecular Probes, Eugene, OR, EUA) for 1 h. Nuclei were counterstained with 10 μL/mL Hoechst 33342 for 20 min. The embryos were washed three times for 10 min in PBS and examined under a fluorescence microscope. Reactions in which the primary antibody was omitted served as negative control. Images of each structure were captured with an AxioCam camera and stored using the AxioVision 4.7.1 software (Carl Zeiss, Jena, Germany).

Images were measured for fluorescence intensity on each blastomere (day 5 embryos) using Adobe Photoshop CS3 (Adobe Systems Inc., Beaverton, OR, USA). First, the three images from each embryo (HOECHST, Alexa 488 and Alexa 555) were placed together in a new file, in different layers. Nuclei were selected with the magic wand tool in HOECHST layer for each blastomere. Sections were measured using the histogram function through the red (H3k27me3) and green (H3k9ac) channels. Photoshop assigns intensity values between 0 and 255 to each pixel in the selected area and then averages these intensities, giving the mean intensity of the selected region. For each embryo, the mean intensity of blastomeres was normalized to the lowest level. After, levels were classified into 7 categories.

Statistical analysis

Mean frequency of each category of normalized blastomeres was analyzed by one-way ANOVA and means were compared by the Tukey test. Statistical analysis was performed using the SAS 9.1 software (SAS Institute Inc., Cary, NC, USA).

Results

We evaluated two replicates and 12 embryos during the transition from 8- to 16-cell stages, totaling 169 blastomeres. The pattern detected for each embryo can be seen in Figure 1. As we can observe, levels of H3k27me3 varied accordingly to levels of H3k9ac. In others words, blastomeres that presented higher H3k27me3 tended to present higher H3k9ac, which means that, for those embryos, global increases on repressive marks (H3k27me3) leads to increases in permissive marks (H3k9ac) as well. Then we normalized the fluorescence level of each blastomere to the lowest level obtained, within each embryo. In this analysis, we observed that some embryos displayed a high individual variation between blastomeres, as demonstrated in Figure 2.

Therefore, we divided the embryos into two classes: A, for embryos that presented similar H3k9ac and H3k27me3 between blastomeres (8 embryos, 66%), and B, for embryos that exhibited variations between blastomeres (at least 2 blastomeres presenting 2fold increase in H3k9ac compared to the lowest blastomere) (4 embryos, 33%). Within each class, we classified the blastomeres accordingly to their intensity level: 1 to 1.5 (I), 1.5 to 2 (II), 2 to 2.5 (III), 2.5 to 3 (IV), 3 to 3.5 (V), 3.5 to 4 (VI), 4 to 4.5 (VII).


Fig. 1. Pattern of H3k9ac and H3k27me3 for 12 different embryos. Rows represent fluorescence levels of individual blastomeres. A) Embryos that presented similar levels of H3k9ac and H3k27me3 between blastomeres. B) Embryos that presented at least 2 blastomeres presenting 2-fold increases on H3k9ac levels, comparing to lowest level.

In class A embryos, we detected for H3k9ac a higher (P < 0.05) frequency of level I blastomeres, corresponding to 80%. The other blastomeres were classified in level II (18.3%) and III (1.6%). In this class of embryos, we did not detect any blastomeres in levels IV-VII, which means that most blastomeres presented similar lower levels of H3k9ac. The same pattern was observed in class I embryos for repressive mark H3k27me3. These results indicate that H3k9ac and H3k27me3 levels are constant between blastomeres in class I embryos, and their variation to the lowest blastomere level is only 2.5 fold maximum, in a small percentage of cells. Also, H3k9ac and H3k27me3 exhibited the same pattern of expression.



[image not available]

Fig. 2. Levels of H3k9ac and H3k27me3 in individual blastomeres from 12- to 16-cell embryos. I) Immunocitochemistry reaction for H3k9ac (Alexa 488, green) and H3k27me3 (Alexa 555, red) in class A (a,b) and class B (d,e) embryos. Nuclei were counterstained with HOECHST 33342 (c,f). II) Percentage (mean ± S.E.) of blastomeres from class A (a,b) and B (c,d) in H3k9ac and H3k27me3 level categories. In each embryo, blastomeres were normalized to the lowest H3k9ac level. Means with different letters within the same group are not equal (ANOVA one way and Tukey post test, P < 0.05).

On the other hand, when we assessed class B embryos, we observed that only 36% blastomeres were classified as level I. This percentage was superior (P < 0.05) when compared to the percentage of embryos classified as levels V (9.2%), VI (1.9%) and VII (1.9%), but was similar to the frequency observed for levels II (14.8%), III (23.2%) and IV (12.4%). Thus, it can be suggested that a higher variation among H3k9ac levels is present in class B embryos. Additionally, for H3k27me3, the same pattern was observed, although level I frequency was higher (48.4%) and similar to level III (18.5%), and both levels I and III were superior (P < 0.05) to levels II (11.7%), IV (5.0%), V (6.6%), VI (3.8%) and VII (5.8%). Based on these results, it can be infered that, for class B embryos, levels of H3k9ac and H3k27me3 displayed a remarkable variation between blastomeres, up to 4.5 fold higher than the lowest blastomere level.
We also wanted, we wanted to confirm if the variations observed for H3k9ac and H3k27me3 were occuring in the same intense for both marks. Therefore, each blastomere level was tested for Person’s correlation analysis. It was observed an extremely high correlation coeficient (r = 0.913) and a significant P value (P < 0.0001), confirming our hypothesis that H3k9ac and H3k27me3 are hightly correlated (Figure 3). In other words, we observed that in 12- to 16-cell embryos repressive and permissive marks vary in the same direction and intensity.


Fig. 3. Correlation between H3k9ac and H3k27me3 in individual blastomeres from 12- to 16-cell embryos.

Conclusions and future directions

Our results describe the presence of two distinguishable populations of bovine embryos during the 4th cell cycle, considering their epigenetic status. One population presented similar levels of repressive and permissive marks in all blastomeres, while the second one displayed a remarkable variation among their blastomeres.

Those changes were reported 2 cell cycles earlier than lineage specification in blastocysts. In mice, it has been demonstrated that differences in histone modifications between blastomeres as early as at the 4-cell stage reflect pluripotency, 2 cycles before trophectoderm differentiation. Furthermore, only a specific population of embryos, based on their cleavage pattern, presented this difference in histone modification levels between blastomeres. Therefore, it is possible that the same phenomena happens in bovine embryos, and that might explain our findings.

In other words, those variable histone modification levels within class B embryos might show differences in pluripotent competence between blastomeres in the same embryo. In this case, cellular differentiation, which can be clearly seen after blastocyst formation, might have already been initiated in the 16-cell bovine embryo. However, this preliminary data should be further investigated. Time lapse studies, following the cell fate decisions of those blastomeres and better characterization of how they were derived are needed to elucidate this question.

In addition, here we demonstrated that global levels of permissive and repressive marks are correlated at this time point. These results suggest that, in 4th cell cycle embryos, no global switch between repressive and permissive marks is detected; and that level of those marks still goes along together.

In this review, embryonic genome activation is a crucial step across early embryo development, and it is accompanied by a dramatic change in epigenetic profile of blastomeres. Monitoring of histone modifications related to euchromatin and heterochromatin is an important tool to assess developmental competence in a sense that those marks are altered when manipulation and environmental stress conditions are applied. We described the pattern of IVC embryos in the cycle after embryonic genome activation, considering a repressive (H3k27me3) and a permissive (H3k9ac) histone modification mark. Those experiments would also be useful in order to compare culture conditions of IVF embryos, and how they would respond after environmental challenges. Interestingly, we found two distinguishable populations of embryos, one presenting similar profiles between blastomeres and other presenting remarkable changes between blastomeres. This observation should be further studied, as it might be reflecting distinct cleavage pattern embryos and pluripotency competence.

In conclusion, epidemiological studies on ART conceived children and molecular analysis in several mammals yield yet contrasting results. The incomplete picture on the safety of ART demands for further studies on the basic biology of fertilization and pre-implantation development. Only a deeper understanding of the cellular and molecular mechanisms ruling life early phases will enable a proper evaluation of the severity of ART impact.

References:

- Bannister, A.J. & Kouzarides, T. (2011). Regulation of chromatin by histone modifications. Cell Research, Vol.21, No.3, (March 2011), pp. 381-395, ISSN 1748-7838
- Maalouf, W.E.; Alberio, R. & Campbell, K.H. (2008). Differential acetylation of histone h4 lysine during development of in vitro fertilized, cloned and parthenogenetically activated bovine embryos. Epigenetics, Vol.3, No.4, (July – August 2008), pp. 199-209, ISSN 1559-2308
- Wolffe, A.P. & Guschin, D. (2000). Review: Chromatin structural features and targets that regulate transcription. Journal of Structural Biology, Vol.129, No.2-3, (April 2000), pp. 102-122, ISSN 1047-8477
- Zhang, J.; Li, X.; Peng, Y.; Guo, X.; Heng, B. & Tong, G. (2010). Reduction in exposure of human embryos outside the incubator enhances embryo quality and blastulation rate. Reproductive Biomedicine Online, Vol.20, No.4, (April 2010), pp. 510-515, ISSN 1472-6483
- http://www.choosingwisely.org/

striker
Posted: Friday, December 12, 2014 1:09:26 PM
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gives some couples hope
Fredric-frank Myers
Posted: Friday, December 12, 2014 3:47:21 PM

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I just ran across this definition that really fits the Christian right: Subphylum 1. subordinate to the phylum 2. superordinate to the superclass. This really covers those that believe this world is only 6000 year's old, that man like us walked with the dinosaur's, or that there was a virgin birth and Jesus was the only child of Mary and never had brothers and sisters. Of course he may have, "arose from the dead", had he not died before being put into a crypt. Marry Christmas to you all out there..
Fredric-frank Myers
Posted: Friday, December 12, 2014 3:47:23 PM

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I just ran across this definition that really fits the Christian right: Subphylum 1. subordinate to the phylum 2. superordinate to the superclass. This really covers those that believe this world is only 6000 year's old, that man like us walked with the dinosaur's, or that there was a virgin birth and Jesus was the only child of Mary and never had brothers and sisters. Of course he may have, "arose from the dead", had he not died before being put into a crypt. Marry Christmas to you all out there..
Fredric-frank Myers
Posted: Friday, December 12, 2014 3:57:00 PM

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Science is so wonderful, but there are those that, for some very demented reasons fight the ability to assist the world, why? They reject vaccinating children, no funding for public schooling but rather home schooling, not allowing a women to protect herself from unwanted pregnancies but then give the,"brain-dead" males Viagra and use insurance to pay for those little blue pills and force the public, during the dinner hour to watch commercial's regarding male erections. Ah yes, this is a great place America.....
monamagda
Posted: Friday, December 12, 2014 6:43:53 PM

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“Biotechnology is assuming a more predominant role in the generation of human offspring at the price of diluting our notion of man as a procreating being.... Man begins to see himself more and more as an individual who stands apart from what he produces, rather than as a being who is created in the image of a Triune God whose inner life is dynamically procreative,”
Technologies of assisted reproduction such as in vitro fertilization (IVF) have been controversial on religious grounds since their inception, but nonetheless, within Islam, Judaism, Confucianism, Hinduism, and most forms of Christianity, adjustments have been made to facilitate the fertility of their adherents [24]. catholicism remains the only major world religion that unequivocally condemns the use of ivf. the vatican argues that the research, development, and practice of ivf involve the destruction of embryos, i.e., the ‘‘destruction of human life,’‘ and by engaging in assisted reproduction, humans are technologically interfering with a process that should remain under god's dominion (ratzinger 1987). to the catholic church, surrogacy; artificial insemination by husband or donor; and ivf are not allowed, because procreation without sexual union in considered unnatural, and the church has been quite vocal about its criticism. church considers in vitro fertilization wrong because it separates human procreation from conjugal union. In the process, couples make themselves the masters of human life instead of its stewards. The strongest protest is on the use of these supernumerary embryos in research which hamper the divinity and dignity of human life.

https://ispub.com/IJH/6/1/4581
Wanderer
Posted: Friday, December 12, 2014 10:37:45 PM

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How expensive is all of this? I had always thought that if I were in the predicament I would adopt. Even after I had children I fostered three. There are so many children that are, for all intents and purposes, thrown away. Just my opinion.
NeuroticHellFem
Posted: Saturday, December 13, 2014 8:06:41 AM

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Wanderer wrote:
How expensive is all of this? I had always thought that if I were in the predicament I would adopt. Even after I had children I fostered three. There are so many children that are, for all intents and purposes, thrown away. Just my opinion.

Good on you, Wanderer. You make a great point. Applause
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