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State of the art in oncologic imaging of breast.

Posted in Bio Instrumentation in Experimental Life Sciences Research, Biomarkers & Medical Diagnostics, CANCER BIOLOGY & Innovations in Cancer Therapy, Computational Biology/Systems and Bioinformatics, Ecosystems & Industrial Concentration in the Medical Device Sector, FDA Regulatory Affairs, Health Economics and Outcomes Research, HealthCare IT, Imaging-based Cancer Patient Management, Medical Devices R&D and Inventions, Medical Devices R&D Investment, Nanotechnology for Drug Delivery, Personalized and Precision Medicine & Genomic Research, Population Health Management, Genetics & Pharmaceutical, Regulated Clinical Trials: Design, Methods, Components and IRB related issues, Reproductive Biology & Bio Instrumentation, Technology Transfer: Biotech and Pharmaceutical, tagged , , , , , , , , , , , , , , , , , , , , on January 21, 2013| 2 Comments »

State of the art in oncologic imaging of breast.

Author-Writer: Dror Nir, PhD

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Word Cloud By Danielle Smolyar

In the coming posts I will address the state of the art in oncologic imaging based on a review paper; Advances in oncologic imaging†‡ that provides updates on the latest approaches to imaging of 5 common cancers: breast, lung, prostate, colorectal cancers, and lymphoma. This paper is published at CA Cancer J Clin 2012. © 2012 American Cancer Society.

The paper gives a fair description of the use of imaging in interventional oncology based on literature review of more than 200 peer-reviewed publications.

In this post I summaries the chapter on breast cancer imaging.

Breast Cancer Imaging

As a start the authors describes the evolution in the ACS imaging guidelines for breast cancer screening. Most interesting to learn is how age limits are changing. The most recent: “In 2010, the Society of Breast Imaging and the Breast Imaging Commission of the ACS issued recommendations for breast cancer screening to provide guidance in light of the controversies and emerging technologies.5 These recommendations were based on multiple prospective randomized trials as well as population-based experience.

Recommendations for screening with non-mammographic imaging are based not on evidence showing mortality reduction but largely on surrogate indicators, i.e., tumor size and nodal status, suggesting improved survival compared with women who are not screened.” I have referred to these guidelines in my recent post: Not applying evidence-based medicine drives up the costs of screening for breast-cancer in the USA.

As long as imaging interpretation is based mainly on observations related to lesion morphology:

“The imaging characteristics of malignant lesions are nonspecific and usually do not allow a definitive diagnosis. When a biopsy is recommended based on mammography, it has a 25% to 45% likelihood of resulting in a diagnosis of carcinoma.11 Similar positive predictive values are reported for biopsies recommended based on MRI.”

It is worthwhile noting that these results do not reflect purely the specificity of the imaging device but rather the specificity of the whole workflow; i.e imaging, biopsy and histopathology. All imaging techniques have false negatives: Mammography screening of general population misses approximately 20% of the cancers. This rate increases as breast density increases. MRI is not applied to general population. When applied to highly suspicious cases MRI misses ~10% of the invasive cancers. Although ultrasound has proven to be useful in detecting cancer especially in women with dense breasts: Automated Breast Ultrasound System (‘ABUS’) for full breast scanning: The beginning of structuring a solution for an acute need! Based on the literature reviewed by the authors of this paper they do not recommend routine sonography for these women.

For women with locally advanced breast cancer (Fig. 2) who undergo neoadjuvant therapy before breast surgery, the authors recommends post-treatment staging using MRI, which has been found to predict complete response with sensitivity above 60% and specificity as high as 90%.26

A 27-year-old female with locally advanced poorly differentiated invasive ductal carcinoma underwent evaluation of extent of disease before starting neoadjuvant chemotherapy. Sagittal fat-suppressed T1-weighted postcontrast MR images demonstrate an almost 6-cm heterogeneously enhancing mass (A) involving the skin of the lower breast (arrow) with (B) right axillary (arrow) and (C) right internal mammary adenopathy (arrow).

A 27-year-old female with locally advanced poorly differentiated invasive ductal carcinoma underwent evaluation of extent of disease before starting neoadjuvant chemotherapy. Sagittal fat-suppressed T1-weighted postcontrast MR images demonstrate an almost 6-cm heterogeneously enhancing mass (A) involving the skin of the lower breast (arrow) with (B) right axillary (arrow) and (C) right internal mammary adenopathy (arrow).

Same is recommended for women who have undergone lumpectomy if the surgical margins are positive. As post therapy follow-up, a new baseline mammogram of the treated breast is recommended followed by annual mammography.

In regards to emerging technology the following are discussed: Mammographic tomosynthesis – see also Improving Mammography-based imaging for better treatment planning

Contrast-enhanced digital mammography – “involves the injection of iodinated contrast material, as is done for computed tomography (CT); this enables hypervascular lesions to be seen with modified mammography technology, potentially providing the same information obtained through MRI. Little has been published on the clinical application of this technology, but diagnostic accuracy better than that of mammography and approaching that of MRI has been reported.31, 32”

MR choline spectroscopy – has been shown to improve the positive predictive value of breast MRI and may be useful in reducing the number of lesions that require biopsy (Fig. 4).33 Studies of spectroscopy have reported sensitivities of 70% to 100% and specificities of 67% to 100% in the detection of breast cancer. Decreasing choline concentrations may also be a useful indication of tumor response to treatment before any change in tumor volume can be detected.34, 35 Technical factors have limited the use of spectroscopy to lesions 1 cm in size or larger.”

Sagittal fat-suppressed T1-weighted postcontrast MR image is shown (A) of the right breast of a 48-year-old female who was status post–contralateral mastectomy for DCIS with the spectroscopy voxel placed over an enhancing mass (arrow). The magnified spectrum (B) demonstrated no choline peak. Biopsy yielded fibroadenoma.

Sagittal fat-suppressed T1-weighted postcontrast MR image is shown (A) of the right breast of a 48-year-old female who was status post–contralateral mastectomy for DCIS with the spectroscopy voxel placed over an enhancing mass (arrow). The magnified spectrum (B) demonstrated no choline peak. Biopsy yielded fibroadenoma.

Diffusion-weighted MRI (DW-MRI) – “adding DW-MRI data to other imaging characteristics of lesions on breast MRI may increase the positive predictive value of the examination, in turn decreasing the number of benign lesions requiring biopsy for diagnosis.” See also Imaging: seeing or imagining? (Part 2).

Axial T1-weighted fat-suppressed postcontrast MR image is shown (A) of the left breast of a 42-year-old female with biopsy-proven contralateral cancer undergoing evaluation of disease extent. An enhancing mass (arrow) was seen in the left breast. This mass (arrow) was also demonstrated on the axial diffusion-weighted MR image (B). Biopsy yielded fibroadenoma with atypical ductal hyperplasia and lobular carcinoma in situ.

Axial T1-weighted fat-suppressed postcontrast MR image is shown (A) of the left breast of a 42-year-old female with biopsy-proven contralateral cancer undergoing evaluation of disease extent. An enhancing mass (arrow) was seen in the left breast. This mass (arrow) was also demonstrated on the axial diffusion-weighted MR image (B). Biopsy yielded fibroadenoma with atypical ductal hyperplasia and lobular carcinoma in situ.

Ultrasound-elastography – “Ultrasound elastography has been reported to differentiate benign from malignant breast lesions with sensitivities of 78% to 100% and specificities of 21% to 98%.39 When added to other US techniques, it may improve radiologists’ performance in distinguishing malignant breast lesions.”

Positron emission tomography (PET) – “alone or combined with CT, allows noninvasive, quantitative assessment of biochemical and functional processes at the molecular level in the body. It is most often performed with the radiolabeled glucose analogue [18F] fluorodeoxyglucose ([18F]FDG) to detect the elevated glucose metabolism that is a hallmark of cancer. In breast cancer, its utility depends on the pretest probability for advanced disease, and thus the clinical stage.” The authors found that the use of [18F] FDG PET to patients with stage I and II disease is “limited”. Specifically, they claim that it is not sufficiently accurate for axillary nodal staging in this subset of patients.40 The did find enough evidence to recommend the use of FDG PET in patients with advanced disease: “where it accurately defines disease extent,41 and frequently eliminates the need for other imaging tests, and provides an early readout of treatment response as well as prognostic information.”

Combined PET/MRI is mentioned as a promising technology for predicting response to therapy “but this remains to be proven”.

Positron emission mammography (PEM) – “adapts full-body PET imaging to the breast. In a multicenter study, the interpretation of PEM in conjunction with mammographic and clinical findings yielded a sensitivity of 91% and a specificity of 93% for breast cancer.47 “. However, the authors mention that its use for screening (applying to healthy women) has been criticized because of the need to administer a radioactive tracer.

Lung Cancer Imaging

To be followed…

Other research papers related to the management of breast cancer were published on this Scientific Web site:

The unfortunate ending of the Tower of Babel construction project and its effect on modern imaging-based cancer patients’ management

 Automated Breast Ultrasound System (‘ABUS’) for full breast scanning: The beginning of structuring a solution for an acute need!

Introducing smart-imaging into radiologists’ daily practice.

Will Bio-Tech make Medical Imaging redundant?

Improving Mammography-based imaging for better treatment planning

Not applying evidence-based medicine drives up the costs of screening for breast-cancer in the USA.

New Imaging device bears a promise for better quality control of breast-cancer lumpectomies – considering the cost impact

Harnessing Personalized Medicine for Cancer Management, Prospects of Prevention and Cure: Opinions of Cancer Scientific Leaders @ http://pharmaceuticalintelligence.com

Predicting Tumor Response, Progression, and Time to Recurrence

“The Molecular pathology of Breast Cancer Progression”

Personalized medicine gearing up to tackle cancer

Whole-body imaging as cancer screening tool; answering an unmet clinical need?

What could transform an underdog into a winner?

Mechanism involved in Breast Cancer Cell Growth: Function in Early Detection & Treatment

Nanotech Therapy for Breast Cancer

A Strategy to Handle the Most Aggressive Breast Cancer: Triple-negative Tumors

Optical Coherent Tomography – emerging technology in cancer patient management

Breakthrough Technique Images Breast Tumors in 3-D With Great Clarity, Reduced Radiation

Closing the Mammography gap

Imaging: seeing or imagining? (Part 1)

Imaging: seeing or imagining? (Part 2)

 

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Oogonial stem cells purified: a view towards the future of reproductive biology

Posted in Biological Networks, Gene Regulation and Evolution, Biomarkers & Medical Diagnostics, Cell Biology, Signaling & Cell Circuits, Medical and Population Genetics, Personalized and Precision Medicine & Genomic Research, Population Health Management, Genetics & Pharmaceutical, Reproductive Andrology, Embryology, Genomic Endocrinology, Preimplantation Genetic Diagnosis and Reproductive Genomics, Reproductive Biology & Bio Instrumentation, Stem Cells for Regenerative Medicine, tagged , , , , on January 14, 2013| 4 Comments »

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

Germline stem cells that produce oocytes in vitro and fertilization-competent eggs in vivo have been identified in and isolated from adult mouse ovaries. A fluorescence-activated cell sorting-based protocol has been standardized that can be used with adult mouse ovaries and human ovarian cortical tissue to purify rare mitotically active cells that have a gene expression profile that is consistent with primitive germ cells. Once established in vitro, these cells can be expanded for months and can spontaneously generate 35- to 50-μm oocytes, as determined by morphology, gene expression and haploid (1n) status. Injection of the human germline cells, engineered to stably express GFP, into human ovarian cortical biopsies leads to formation of follicles containing GFP-positive oocytes 1–2 weeks after xenotransplantation into immunodeficient female mice. Therefore, based on the multiple experimental lines of evidence reported it is reasonable to conclude that the rare cells with cell-surface expression of DDX4 that are present in the ovaries of reproductive-age women represent adult human OSCs. In addition to opening a new research field in human reproductive biology that was inconceivable less than 10 years ago, clear evidence for the existence of these cells in women may offer new opportunities to expand on and enhance current fertility-preservation strategies. For example, with assisted reproductive technologies involving cryopreservation of ovarian cortical tissue already in development for females with cancer, isolation and expansion of OSCs from this tissue before or after cryopreservation might be useful for new fertility applications. In fact, it has been found that these cells can be consistently obtained from cryopreserved and thawed human ovarian tissue samples, and that these cells per se can be cryopreserved and thawed months later with minimal loss for successful establishment in vitro. In addition, the availability of a detailed protocol for the purification of these newly discovered cells from human ovary tissue provides a much more physiologically relevant in-vitro model system from which to study human female germ cell development compared to the ESC-derived or induced pluripotent stem cell-derived germline cells that are currently used as models for human female gametogenesis.

Source References:

http://blogs.nature.com/spoonful/2012/02/video-stem-cell-discovery-puts-women%E2%80%99s-reproduction-on-fertile-grounds.html

http://www.nature.com/nm/journal/v18/n3/full/nm.2669.html

http://www.ncbi.nlm.nih.gov/pubmed/23024060

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Hypogonadotropic hypogonadism

Posted in Reproductive Andrology, Embryology, Genomic Endocrinology, Preimplantation Genetic Diagnosis and Reproductive Genomics, Reproductive Biology & Bio Instrumentation, tagged , , , , on January 7, 2013| 1 Comment »

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

 

Hypogonadotropic hypogonadism is a form of hypogonadism that is due to a problem with the pituitary gland or hypothalamus. In this condition, the male testes or the female ovaries produce little or no hormones.

 

Causes:

Hypogonadotropic hypogonadism is caused by a lack of secretion of the gonadal stimulating pituitary hormones: follicle stimulating hormone (FSH) and luteinizing hormone (LH). Normally, the hypothalamus in the brain releases gonadotropin-releasing hormone (GnRH). This hormone stimulates the pituitary gland to release other hormones, including FSH and LH. These hormones tell the female ovaries and male testes to release hormones that lead to normal sexual development in puberty. Any change in this hormone release chain causes a lack of sex hormones and prevents normal sexual maturity. Failure of the hypothalamus is usually a result of Kallmann syndrome. Kallmann syndrome is an inherited form of hypogonadotropic hypogonadism that can occur with a loss of smell.

Symptoms:

  • Erectile dysfunction in men
  • Inability to smell (in some cases)
  • Lack of development at puberty (development may be incomplete or delayed)
  • Lack of secondary sexual characteristics such as pubic, facial, and underarm hair
  • Loss of menstrual periods in women
  • Short stature (in some cases)
  • Underdeveloped testicles

 

Treatment:

Treatment depends on the source of the problem, but may involve:

  • Estrogen and progesterone pills
  • GnRH injections
  • Injections of testosterone
  • Slow-release testosterone skin patch
  • Surgery to remove a pituitary tumor
  • Testosterone gels

 

Expectations (prognosis):

With the right hormone treatment, the person can go through puberty and fertility may be restored.

 

Complications:

  • Delayed puberty
  • Infertility
  • Low self-esteem due to late start of puberty (emotional support may be helpful)
  • Sexual dysfunction

 

Prevention:

Prevention depends on the cause. People who have a family history of inherited conditions that cause hypogonadism may benefit from genetic counseling. Preventing serious head injuries reduces the risk of hypogonadotropic hypogonadism due to pituitary injury.

 

Source References:

 

http://www.umm.edu/ency/article/000390.htm

 

http://health.nytimes.com/health/guides/disease/hypogonadotropic-hypogonadism/overview.html

 

http://www.ncbi.nlm.nih.gov/books/NBK1278/

 

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Fallopian tube cilia – its reproductive significance

Posted in Reproductive Biology & Bio Instrumentation, tagged , , , , , , , on December 30, 2012| 1 Comment »

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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Women’s Health and the Human Pheromone

Posted in Bio Instrumentation in Experimental Life Sciences Research, Cell Biology, Signaling & Cell Circuits, Molecular Genetics & Pharmaceutical, Personalized and Precision Medicine & Genomic Research, Reproductive Andrology, Embryology, Genomic Endocrinology, Preimplantation Genetic Diagnosis and Reproductive Genomics, Reproductive Biology & Bio Instrumentation, Technology Transfer: Biotech and Pharmaceutical, tagged , , , , , , , , , on December 21, 2012| 1 Comment »

Women

 

Author: Jukka Karjalainen, MD, PhD

 

Sorry ladies, you will be seduced, pheromones make it impossible for you to resist men, no matter how dreadful the man wearing the pheromones may be.

Wait, please don’t panic. Sadly, the pheromone marketing craze may be causing us to turn a blind eye to an interesting discovery. As far as I see it’s like hearing about vitamins for the first time from a hard core drug dealer. When you get over your encounter with Mr. Dealer, you are not going to think of vitamins in the same way as a person who had heard about vitamins from GNC or Vitamin World. I believe the same thing is happening with marketers and pheromones. With that in mind let’s take a deeper look at pheromones.
Most people still believe pheromones are no different from X-ray glasses sold in the back of comic books. Some have been using them for years. To be sure, they are used heavily by government agencies worldwide. Business uses them daily, you may even use them. Of course I’m talking about insect and animal pheromones.
It was well known by the late 70s that females of the insect and animal kingdom produced chemicals for attracting males of the same species. Several examples were presented in literature. By the late 70s pheromones were already being manufactured for pest control. Indeed, pheromones were being used to attract or repel bugs and animals. Pheromones were already protecting crops from damage. Roaches were checking in and not checking out. At the same time scientist were working hard to find and prove the existence of human pheromones. This evidence was found in the mid 70s but did not reach the public with any power until the mid 80s.

Human pheromones made front page news in 1986 when Researchers at the Monell Chemical Senses Center of Philadelphia released their findings to the scientific journal Hormones and Behavior, as well as to the public by way of:

  • Time Magazine: “Studies find that male pheromones are good for women’s health.”
  • News week: “The Chemistry Between People: Are Our Bodies Affected by Another Person’s Scent?”
  • USA Today: “The Real Chemical Reaction between the Sexes.”
  • The Washington post: “Pheromones Discovered in Humans.”

The human pheromone was big news in the 80s. It was found that women’s health was directly affected by male pheromone. Interestingly, Monell Chemical Senses Center of Philadelphia reported that women who work or live together tend to get their menstrual cycles in sync. That curious phenomenon known for years by scientists and many ordinary folk, has long been suspected as an indication that humans, like insects and some mammals, communicate subtly by sexual aromas known as pheromones. (1)

In 1986 Dr. Winnifred Cutler, a biologist and behavioral endocrinologist, co discovered pheromones in our underarms. She and her team of researchers found that once any overbearing underarm sweat was removed, what remained were the odorless materials containing the pheromones. The approach to test the hypothesis was interesting: women and men emitted pheromones into the atmosphere and the authors showed that extracted pheromones could be collected, frozen for over a year, thawed and then applied topically above the upper lip of recipients to mimic some of the pheromonal effects found in nature. Dr. Cutler’s original studies in the ’70s showed that women who have regular sex with men have more regular menstrual cycles than women who have sporadic sex. Regular sex delayed the decline of estrogen and made women more fertile. This led the research team to look for what the man was providing in the equation. By 1986 they realized it was pheromones. (1, 2, 3).
Male scents play a role in maintaining the health of women, particularly the health of the female reproductive system. Pheromones help to maintain the health of women. To be more exact, they keep a woman’s reproductive system healthy. They found that women who have sex with men at least once a week are more likely to have normal menstrual cycles, fewer infertility problems and a milder menopause than celibate women and women who have sex rarely or sporadically. A healthy testosterone rich male pheromone signature somehow encouraged a woman’s body to keep itself healthy and young.

The scent of a good man may be music to a woman’s nose. Researchers also found that exposure to the male pheromones also prompted a shift in blood levels of a reproductive hormone called luteinizing hormone (LH). Levels of this hormone typically surge before ovulation, but women also experience small surges during other times in the menstrual cycle. It also can stabilize the menstrual cycle and reduce the symptoms of PMS. Pheromones could lift a woman’s mood actually alleviating depression, even postpone and then alleviate menopause health. (1, 2)
How did we get from health benefits to wild seduction products? People can’t resist a fast buck. If it’s about money, maybe we should be using pheromone products to make women’s lives better. Strike that. We should instead be using pheromone products to make people’s lives better. Provide pheromones that do the things mentioned above. Help to enable pheromone research that will gain more knowledge related to health and longevity. I don’t have anything against attracting the opposite sex. I think that’s a good idea. It’s just sad to see a good thing, or potentially good thing, be lost because of a poorly focus on health.

There is always more to the story than meets the eye. The person who does not ask questions has either been beaten down low by the people who know-it-all, or, they are the people who know-it-all. Keep asking questions. You will keep finding better answers.

REFERENCES: 
1. Biology of Reproduction, June 2003. News release, University of Pennsylvania.
2. Cutler WB, Preti G, Krieger A, Huggins GR, Garcia GR, Lawley HJ. Human axillary secretions influence women’s menstrual cycles: the role of donor extract of men. Horm Behav 1986; 20: 463473.
3. McCoy and Pitino. Pheromonal influences on sociosexual behavior in young women. Physiology & Behavior 2002; 75: 367-375.

 

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The FEMALE reproductive system and the hypothalamic-pituitary-thyroid axis

Posted in Population Health Management, Genetics & Pharmaceutical, Reproductive Andrology, Embryology, Genomic Endocrinology, Preimplantation Genetic Diagnosis and Reproductive Genomics, Reproductive Biology & Bio Instrumentation, tagged , , , , , , on December 11, 2012| 2 Comments »

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

Increasing evidence derived from experimental and clinical studies suggests that the hypothalamic–pituitary–thyroid axis (HPT) and the hypothalamic–pitutitary–ovarian axis (HPO) are physiologically related and act together as a unified system in a number of pathological conditions. The suggestion that specific thyroid hormone receptors at the ovarian level might regulate reproductive function, as well as the suggested influence of estrogens at the higher levels of the HPT axis, seems to integrate the reciprocal relationship of these two major endocrine axes. Both hyper- and hypothyroidism may result in menstrual disturbances. In hyperthyroidism the most common manifestation is simple oligomenorrhea. Anovulatory cycles are very common. Increased bleeding may also occur, but it is rare. Hypothyroidism in girls can cause alterations in the pubertal process; this is usually a delay, but occasionally it can result in pseudoprecocious puberty. In mature women hypothyroidism usually is associated with abnormal menstrual cycles characterized mainly by polymenorrhea, especially anovulatory cycles, and an increase in fetal wastage.

Thyrotoxicosis and the female reproductive system
Increased sex hormone-binding globulin (SHBG) concentration is characteristic of hyperthyroidism so much so that this globulin is used as a test of thyroid function, reflecting the tissue response to the thyroid hormones. Serum levels of estradiol and testosterone should thus be interpreted with this fact in mind because their total amounts are increased out of proportion to the free levels. Also, women with thyrotoxicosis have a decrease in the metabolic clearance rates of testosterone and of estradiol, an increase in the 5α/5β-reduced metabolites in the urine, and an increase in catechol estrogens in the urine at the expense of estriol and other 16-hydroxylated estrogen metabolites. Southern et al. have found an increase in the peripheral aromatization of androgens to estrogens in some thyrotoxic women. Other studies, however, did not confirm these findings and suggested that any increase in the peripheral aromatization of androgens is likely to be due to an increase in the peripheral blood flow and and not to the direct effect of T4 on the aromatase complex. One of the earliest clinical changes observed in thyrotoxicosis was the occurrence of amenorrhea, which was first reported in 1840. Amenorrhea has been reported frequently since then, but a number of other changes in menstrual cycles have been noted, including anovulation, oligomenorrhea, and menometrorrhagia, which is more common in hypothyroidism. Whether these changes are due to a direct action of T4 on the ovary and uterus or on the pituitary and hypothalamus or both is uncertain. The effect of T4 on fertility is less well established, although the disturbances in menstrual cycles will obviously disturb fertility. With therapy, the menstrual cycles return to their regular pattern for the individual. In summary, thyrotoxicosis occurring in prepubertal girls may result in slightly delayed menarche. In adult women, the effects of thyrotoxicosis on the reproductive system are seen on the hypothalamic–pituitary axis with alterations in gonadotropin release and also in the circulating levels of SHBG, which alter steroid metabolism or biologic activity. These effects produce the variable clinical picture seen in women with thyrotoxicosis.

Hyperthyroidism and physical development
Children born with neonatal Graves’ disease have no defects in the reproductive system that can be related to this disease. Physical development is normal, however, and skeletal growth is often accelerated without a modification of final height. The delay in puberty may be related to the impact of thyrotoxicosis on body composition (i.e., decreased percentage of body fat), which is thought to be related to the onset of puberty and menarche. Rarely, thyrotoxicosis may occur in children in association with polyostotic fibrous dysplasia, cafe au lait pigmentation, and precocious puberty (McCune-Albright syndrome). Precosity has been described in one-third of the affected girls; and large, unilateral follicular cysts may be present and presumably are responsible for the sexual precocity of the gonadotropin-independent type. Thyrotoxicosis results from single or multiple thyroid adenomas.

Hyperthyroidism and menstrual cycle
Thyrotoxicosis occurring before puberty has been reported to delay sexual maturation and the onset of menses, although Saxena did note that, in thyrotoxic girls, the mean age of menarche was slightly advanced over that of their control population without endocrine disease. The association of thyrotoxicosis and precocity in this disorder appears to be coincidental. Although ovulatory menstrual cycles occur in women with thyrotoxicosis, menstrual disturbances are common. Therefore, the possibility of pregnancy should be considered in an amenorrheic thyrotoxic woman. This is important because many of these women may receive treatment with radioactive iodine, which is contraindicated in pregnancy. Oligomenorrhea is the most common abnormality, and it may progress to amenorrhea, first described in the classic study by von Basedow. Polymenorrhea is distinctly less frequent, in comparison to its occurrence in hypothyroidism. The frequency of these menstrual disorders varies in different series. Benson and Dailey found that out of 221 hyperthyroid patients 58% had oligomenorrhea or amenorrhea and 5% polymenorrhea. This is in general agreement with other older studies such as those of Goldsmith et al. More recently, in India Joshi et al. found menstrual irregularities in 64.7% of hyperthyroid women, compared to 17.2% of healthy controls. These irregularities sometimes preceded thyroid dysfunction. However, Krassas et al. found irregular cycles in only 21.5% out of 214 thyrotoxic patients. These discrepant results may be attributed to either genetic and other factors or to more delayed diagnosis in India than in Greece. The weight loss and psychologic disturbances (primarily anxiety) seen in thyrotoxicosis may also contribute to sexual dysfunction. Thyrotoxic women frequently present with increased LH, FSH, and estrogen levels; and the gonadotropin response to gonadotropin releasing hormone (GnRH) is increased, although the mid-cycle LH peak may be reduced or absent.

Hypothyroidism and the female reproductive system
Production of SHBG is decreased in hypothyroidism. As a result, serum estradiol and testosterone concentrations are reduced, although free levels of these hormones remain normal. The metabolism of both androgens and estrogens is also altered in hypothyroidism. Androgen secretion is decreased, and the metabolic transformation of testosterone shifts toward androstenedione rather than androsterone, the reverse of that seen in hyperthyroidism. With respect to estradiol and estrone, hypothyroidism favors metabolism of these steroids via 16α-hydroxylation over 2-oxygenation, resulting in increased formation of estriol at the expense of 2-hydroxyestrone and its derivative, 2-methohyestrone. The alterations in steroid metabolism disappear when the euthyroid state is restored. Serum FSH and LH values are usually normal, but the midcycle FSH and LH surge may be blunted or absent. In postmenopausal women, serum FSH and LH concentrations may be somewhat lower than expected, and the response to GnRH may be reduced. The anovulation is reflected in the frequent finding of aproliferative endometrium on endometrial biopsy. TRα-1 and TRβ-1 receptors have been found in follicular fluid. Earlier work indicated that thyroxine enhanced the action of gonadotropins on luteinization and progestin secretion by cultured granulosa cells, and it has been recently noted that in a group of infertile women, those with elevated TSH levels had a higher incidence of out-of-phase biopsies than women with normal TSH. The defects in hemostasis reported in hypothyroidism, such as decreased levels of factors VII, VIII, IX, and XI, may also contribute to the pathogenesis of polymenorrhea. Ovulation and conception can occur in mild hypothyroidism, but in the past those pregnancies that did occur were often associated with abortions in the first trimester, stillbirths, or prematurity. Recent studies indicate these events may be less common but that gestational hypertension occurs often in pregnant women with untreated hypothyroidism. Pregnancy occurring in women with myxedema has been reported to be uncommon, but this is somewhat hard to document and may be the result of anovulation. The use of L-thyroxine is not helpful in treating euthyroid patients for infertility, menstrual irregularity, or the premenstrual syndrome. Some myxedematous women will present with amenorrhea and galactorrhea and elevated serum prolactin concentrations. Thus, thyroid evaluation should be an essential part of the work-up in any person with galactorrhea. If hypothyroidism is the cause, the amenorrhea and galactorrhea and elevated serum prolactin will disappear promptly with thyroxine therapy. There is an increased incidence of Hashimoto’s thyroiditis in individuals with Turner’s syndrome, and, although a chromosomal linkage between autoimmune disease and the X chromosome has been suggested, this has not been confirmed. Inherited abnormalities in serum TBG are X-linked, and patients with Turner’s syndrome may have low serum TBG values. Women with hypothyroidism have decreased metabolic clearance rates of androstenedione and estrone and increased peripheral aromatization. The ratio of 5α/5β metabolites of androgens is decreased in hypothyroid women, and there is an increase in the excretion of estriol and a decrease in the excretion of 2-hydroxyestrone and its derivative 2-methoxyestrone.

Hypothyroidism and physical development
The reproductive tract appears to develop normally in cretins; thus, hypothyroidism during fetal life does not appear to affect the normal development of the reproductive tract. Hypothyroidism in prepubertal years generally leads to short stature and may lead to a delay in sexual maturity.

Hypothyroidism and the menstrual cycle
An interesting syndrome described by Kendle and Van Wyk and Grumbach occurs not infrequently: it is characterized by precocious menstruation, galactorrhea, and sella enlargement in girls with juvenile hypothyroidism. The cause is thought to be an overlap in the pituitary production of TSH and gonadotropins, with the latter causing early ovarian secretion of estrogens and subsequent endometrial stimulation with vaginal bleeding. Prolactin levels are elevated, leading to galactorrhea. The estrogen and progesterone response of the ovary to human chorionic gonadotropin is increased, possibly from prolactin induction of ovarian LH receptors. In this way hyperprolactinemia may sensitize the ovaries to the low circulating gonadotropin levels present prepubertally. However, there is no pubertal increase in the adrenal production of androgen precursors, so that axillary and pubic hair are usually not apparent. Therapy with thyroxine in proper dosage results in prompt alleviation of the symptoms. In adult women, hypothyroidism results in changes in cycle length and amount of bleeding and has been reported in association with the ovarian hyperstimulation syndrome.108 In an Indian study, 68.2% of hypothyroid women had menstrual abnormalities, compared to 12.2% of healthy controls. Menorrhagia is a frequent complaint and is probably due to estrogen breakthrough bleeding secondary to anovulation, which is frequent in severe hypothyroidism. Some investigators have reported a high incidence of thyroid hypofunction in women with premenstrual syndrome. Most of the thyroid disease was subclinical hypothyroidism, defined as an augmented response of TSH to TRH. Many of the affected women were reported to have complete relief of premenstrual syndrome (PMS) symptoms with L-T4 therapy.

Source References:

http://www.ncbi.nlm.nih.gov/pubmed/10818393

 

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FDA Guidelines For Developmental and Reproductive Toxicology (DART) Studies for Small Molecules

Posted in Bio Instrumentation in Experimental Life Sciences Research, Biomarkers & Medical Diagnostics, BioSimilars, Chemical Biology and its relations to Metabolic Disease, Disease Biology, Small Molecules in Development of Therapeutic Drugs, FDA Regulatory Affairs, Health Economics and Outcomes Research, Health Law & Patient Safety, Pharmaceutical Industry Competitive Intelligence, Population Health Management, Genetics & Pharmaceutical, Regulated Clinical Trials: Design, Methods, Components and IRB related issues, Reproductive Biology & Bio Instrumentation, Uncategorized, tagged , , , , , , , , , , , , , on November 20, 2012| 3 Comments »

FDA Guidelines For Developmental and Reproductive Toxicology (DART) Studies for Small Molecules. Author-Writer: Stephen J. Williams, Ph.D.

This posting is a follow-up on the Report on the Fall Mid-Atlantic Society of Toxicology Meeting “Reproductive Toxicology of Biologics: Challenges and Considerations post and gives a brief synopsis of the current state of FDA regulatory guidelines with respect to DART studies on small molecule (non-biological based) therapeutics.    The following is adapted from the book Principles and Methods of Toxicology by Dr. A Wallace Hayes (1) and is an excellent reference on reproductive toxicology and testing methods.

Chemical insult occurs to the human reproductive system at a multitude of stages in development and the life cycle, leading to the extensive testing which must be performed to diligently the reproductive and development toxicity of a chemical/drug.  Abnormalities and toxic manifestations in the offspring may result from insult to the adult reproductive (either female or male) and neuroendocrine systems, as well as damage to the embryo resulting in embryolethality, fetus at any period during organogenesis, juvenile development or, in the case of certain antibody therapies, immune system development.  The latter, toxic insult to the developing immune system could possibly be manifested as either an immune defect in the newborn or, later in life, as tolerance to said therapy.  It is estimated that exposure to the pregnant woman, of either environmental contaminants or drug, is significant.  It is estimated that a mother may be taking an average of 8-9 different drug preparations, mostly over the counter preparations such as antacids, vitamin preparations, cathartics etc. with the maximal drug intake occurring between 24 and 36 weeks of gestation.

Toxic insult to the developing embryo is dependent on

  • Fetal development stage during drug/chemical exposure
  • Maternal/placental xenobiotic metabolism
  • Pharmacokinetic parameters affecting bioavailability and fetal/maternal drug binding

The following table shows the dependency of developmental stage to teratogenicity: adapted from J. Manson, H. Zenick, and R.D. Costlow from Principles and Methods of Toxicology.

Developmental Stage Major Susceptibility
Preimplantation Embryolethality
Organogenesis Births defects; embryolethality
Fetal Growth retardation, fetal death, functional deficits
Neonatal Growth retardation, nervous system alterations, immune and endocrine systems

It is not generally accepted that there is a dose dependency of teratogenesis however most teratogens have specific mechanisms of action and teratogenic effects occur at much lower doses than result in maternal toxicity.   However, the developmental toxicity may be manifested later in life, including as reproductive toxicity affecting adult fertility and familial generations.

FDA Guidelines for DART Studies on Non-Biologics (Small Molecule Therapeutics)

The basic design for DART studies incorporate the aforementioned principles of tetralogy:

  • developmental stage of fetal exposure
  • parental effects on reproduction and development
  • toxicity may be manifested over multiple generations including fertility rates

Therefore two designs are generally used for DART studies

  1. exposure across several generations
  2. exposure during one generation

FDA requires one control group and two treatment groups, and evaluation of at least two species.  However, most studies will use two rodent and one nonrodent species.

Multigenerational Design

Multigenerational DART studies are conducted for compounds likely to concentrate in the body following long-term exposure.  Examples of types of compounds include pesticides and food additives.

Figure 1.  General Design of a Multigenerational DART study.  Weanlings (30-30 days of age) from the parental generation are treated for a period up to 60 days. At 100-120 days of parental generation, animals are mated.  Fx = filialx .

Three Segment, Single Generation Tests

The single generation design is more suitable for DART studies on drugs, as most therapeutic would be taken over short periods (during pregnancy) and have relatively short half-lives in the body.  FDA guidelines separate these studies in three phases:

I.            Phase I: evaluation of fertility and general reproductive performance

II.            Phase II: assessment of teratogenicity and embryotoxicity

III.            Phase III: peri- and postnatal evaluations.

All figures are adapted from Principles and Methods of Toxicology.(1)

FDA guidelines Guidance for Industry Reproductive and Developmental Toxicities —Integrating Study Results to Assess Concerns can be found at: http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm079240.pdf

FDA Guideline for reproductive toxicity testing for small molecule therapeutics can be found at:

http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm074950.pdf

1.            Hayes, A. W. (1986) Principles and Methods of Toxicology, Raven Press, New York

Other research papers on Pharmaceutical Intelligence and Reproductive Biology, Bio Insrumentation, Endocrinology Genetics were published on this Scientific Web site as follows

Non-small Cell Lung Cancer drugs – where does the Future lie?

Reboot evidence-based medicine and reconsider the randomized, placebo-controlled clinical trial

Every sperm is sacred: Sequencing DNA from individual cells vs “humans as a whole.”

Leptin and Puberty

Gene Trap Mutagenesis in Reproductive Research

Genes involved in Male Fertility and Sperm-egg Binding

Hope for Male Contraception: A small molecule that inhibits a protein important for chromatin organization can cause reversible sterility in male mice

Pregnancy with a Leptin-Receptor Mutation

The contribution of comparative genomic hybridization in reproductive medicine

Sperm collide and crawl the walls in chaotic journey to the ovum

Impact of evolutionary selection on functional regions: The imprint of evolutionary selection on ENCODE regulatory elements is manifested between species and within human populations

Biosimilars: CMC Issues and Regulatory Requirements

Biosimilars: Intellectual Property Creation and Protection by Pioneer and by Biosimilar Manufacturers

Assisted Reproductive Technology Cycles and Cumulative Birth Rates

Innovations in Bio instrumentation in Reproductive Clinical and Male Fertility Labs in the US

Increased risks of obesity and cancer, Decreased risk of type 2 diabetes: The role of Tumor-suppressor phosphatase and tensin homologue (PTEN)

Guidelines for the welfare and use of animals in cancer research

Every sperm is sacred: Sequencing DNA from individual cells vs “humans as a whole.”

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Every sperm is sacred: Sequencing DNA from individual cells vs “humans as a whole.”

Posted in Biological Networks, Gene Regulation and Evolution, Biomarkers & Medical Diagnostics, Chemical Genetics, Genome Biology, Genomic Testing: Methodology for Diagnosis, Medical and Population Genetics, Molecular Genetics & Pharmaceutical, Personalized and Precision Medicine & Genomic Research, Pharmaceutical R&D Investment, Population Health Management, Genetics & Pharmaceutical, Proteomics, Reproductive Andrology, Embryology, Genomic Endocrinology, Preimplantation Genetic Diagnosis and Reproductive Genomics, Reproductive Biology & Bio Instrumentation, Statistical Methods for Research Evaluation, Technology Transfer: Biotech and Pharmaceutical, tagged , on November 5, 2012| 5 Comments »

Reporter: Aviva Lev-Ari, PhD, RN

Genomics: The single life

Sequencing DNA from individual cells is changing the way that researchers think of humans as a whole.

31 October 2012

The tendency of sperm to swim alone makes the cells ideal for single-cell genomics. Adam Auton, a statistical geneticist at Albert Einstein College of Medicine in New York is using sperm to study recombination, the process that shuffles genes during the formation of germ cells and therefore influences which genes are inherited. “Recombination is one of the fundamental forces that shapes genetic diversity,” he says. “In recent years we’ve learned that there is considerable variation in the recombination rate between different populations, between the sexes and even between individuals.” But pinning down the rate in people once seemed impossible because it would have required finding individuals with hundreds of children and sequencing their genomes.

The ability to sequence single cells meant that researchers could take another approach. Working with a team at the Chinese sequencing powerhouse BGI, Auton sequenced nearly 200 sperm cells and was able to estimate the recombination rate for the man who had donated them. The work is not yet published, but Auton says that the group found an average of 24.5 recombination events per sperm cell, which is in line with estimates from indirect experiments2. Stephen Quake, a bioengineer at Stanford University in California, has performed similar experiments in 100 sperm cells and identified several places in the genome in which recombination is more likely to occur. The location of these recombination ‘hotspots’ could help population biologists to map the position of genetic variants associated with disease.

Quake also sequenced half a dozen of those 100 sperm in greater depth, and was able to determine the rate at which new mutations arise: about 30 mutations per billion bases per generation3, which is slightly higher than what others have found. “It’s basically the population biology of a sperm sample,” Quake says, and it will allow researchers to study meiosis and recombination in greater detail.

SOURCE:  

VIEW ARTICLE IN NATURE

http://www.nature.com/news/genomics-the-single-life-1.11710#/genome

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Leptin and Puberty

Posted in Population Health Management, Genetics & Pharmaceutical, Reproductive Andrology, Embryology, Genomic Endocrinology, Preimplantation Genetic Diagnosis and Reproductive Genomics, Reproductive Biology & Bio Instrumentation, tagged , , , , , , , on November 5, 2012| 5 Comments »

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

It is well established that food restriction delays pubertal onset, whereas refeeding abolishes this delay. In addition, murine and human genetic models of leptin deficiency fail to enter puberty, and treatment with leptin can establish a pulsatile secretory pattern of gonadotropins that is characteristic of early puberty. The female transgenic skinny mouse, which is an in vivo model of chronic hyperleptinemia in the absence of adipose tissue, enters puberty precociously. Data regarding the effects of leptin administration on pubertal onset are controversial. It has been shown that intracerebroventricular leptin administration prevents the delay in vaginal opening induced by chronic food restriction in the rat. By contrast, it has been found that artificially raised leptin levels are not sufficient to abolish the delay of pubertal onset caused by food deprivation. Thus, the question arises whether leptin might be a ‘permissive factor’ (tonic mediator), whose concentration above a certain threshold is required for pubertal onset, or a ‘trigger’ (phasic mediator) that determines the pubertal spurt through a rise in serum concentration at an appropriate time of development.

The temporal correlation between increases in leptin concentration and the initiation of LH pulsatility over the peripubertal period has been studied in several species. In men it has been shown that leptin levels rise by 50% before the onset of puberty, and decrease to baseline after the initiation of puberty. Other cross-sectional studies showed that age has a significant effect on serum leptin concentrations through prepuberty into early puberty. It has been reported repeatedly that there are no significant changes in leptin levels over the peripubertal period in male rhesus macaques; however, more recent studies performed in castrated male monkeys showed that nocturnal levels of leptin increase just before the nocturnal prepubertal increase in pulsatile LH release.

A possible explanation for such contrasting reports in monkeys could be the sampling of nocturnal rather than diurnal blood. Indeed, in primates, prepubertal changes in nocturnal LH release occur approximately five months before diurnal variations. Another reason might be the use of different models: agonadal monkeys were treated with intermittent exogenous GnRH to sensitize the pituitary to endogenous GnRH, thus magnifying the LH release independently from gonadal influences. In the same study, the leptin rise was accompanied by a sustained increase in nocturnal GH and IGF-I concentrations before the onset of puberty, which is defined as the increase in nocturnal pulsatile LH secretion. It is not clear whether one of the two metabolic signals has a predominant role or whether both act in concert. Indeed, it has been reported that the maximum increase in GH and leptin occurs simultaneously, about 10–30 days before the onset of puberty. However, these conclusions were based on results from a study that used castrated animals, which in the strictest sense do not undergo puberty. Thus, it remains to be clarified whether the same mechanisms that result in the onset of the pubertal rise in LH secretion in castrated animals are also responsible for the reactivation of the HPG axis in intact animals.

The sexual dimorphism in leptin concentrations becomes evident after puberty. In males, leptin levels rise throughout childhood, reach a peak in the early stages of puberty and then decline, whereas they increase steadily during pubertal development in females. Consequently, leptin levels are three to four times higher in females than in males. The reason for this postpubertal sexual dimorphism in leptin levels is not clear. After puberty, serum testosterone and testicular volume are inversely related to leptin levels in males, whereas in females, when adjusted for adiposity indexes, estradiol is directly correlated with leptin levels. These observations indicate that androgens and estradiol might account, at least in part, for the gender differences in circulating leptin levels. This is also supported by in vitro studies which show that androgens and estrogens inhibit and stimulate leptin expression and release from human adipocytes in culture, respectively.

Thus, puberty represents a turning point in the sexual dimorphic relationships between the HPG axis and leptin by determining the steroid milieu that leads to a different regulation of leptin secretion in the sexes.

Source References:

http://www.sciencedirect.com/science/article/pii/S1043276000003520#

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Gene Trap Mutagenesis in Reproductive Research

Posted in Bio Instrumentation in Experimental Life Sciences Research, Biological Networks, Gene Regulation and Evolution, Biomarkers & Medical Diagnostics, Cell Biology, Signaling & Cell Circuits, Chemical Genetics, Computational Biology/Systems and Bioinformatics, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Genome Biology, Genomic Testing: Methodology for Diagnosis, Medical and Population Genetics, Molecular Genetics & Pharmaceutical, Personalized and Precision Medicine & Genomic Research, Pharmaceutical Analytics, Pharmaceutical Industry Competitive Intelligence, Pharmaceutical R&D Investment, Population Health Management, Genetics & Pharmaceutical, Proteomics, Reproductive Andrology, Embryology, Genomic Endocrinology, Preimplantation Genetic Diagnosis and Reproductive Genomics, Reproductive Biology & Bio Instrumentation, Stem Cells for Regenerative Medicine, Technology Transfer: Biotech and Pharmaceutical, tagged , , , , , , , , , , on October 18, 2012| 3 Comments »

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

With the completion of the mapping of the human genome, we now have access to all the DNA sequence information responsible for human biology. Together with microarray technology, we are ushering in a new era in reproductive medicine—the era of Reproductive Genomics.

Whole genome microarray analysis of the testis and ovary suggests that a substantial part of the genome is expressed in reproductive tissues and many of them are likely to be important for normal reproduction. Yet adequate expression and functional information is only available for less than 10% of them. Hence, one of the important questions in reproductive studies now is ‘how do we associate function with the genes expressed in reproductive tissues?’ The establishment of mutations in animal models such as the mouse represents one powerful approach to address this question.

Animal models have played critical roles in improving our understanding of mechanisms and pathogenesis of diseases. Mouse knockout models have often provided highly needed functional validation of genes implicated in human diseases. The rapid advance of human genetics in areas such as

  • single nucleotide polymorphisms (SNP) and
  • haplotyping technology

now allows the identification of disease-associated single nucleotide variation at a much faster pace. Functional examination of those candidate genes is needed to determine if those genes or variants are indeed involved in reproductive disease. Generating mutations in murine homologs of candidate genes represents a direct way to determine their roles, and mouse models will further allow the dissection of genetic pathways underlying the disease condition and provide models to test possible drug treatments. Thus, how to generate mouse models efficiently becomes a priority issue in the Genomics era of Reproductive Medicine.

It is known that generating a mouse knockout is no small endeavor, even for a mouse research lab, often requiring specialized expertise and experience in

  • molecular biology,
  • embryonic stem (ES) biology and
  • mouse husbandry.

Therefore, it could be intimidating for people who have little experience in mouse research. Fortunately, there are some technological developments in the mouse community that make the task of generating mouse mutations less intimidating to people unfamiliar with mouse genetics. One of these developments is the effort led by the International Gene Trap Consortium (IGTC) to generate a library of mouse mutant ES cells covering most of the genes in the mouse genome. This method saves researchers the tedious and sometimes challenging tasks of making knockout vectors and screening ES cell colonies and directly provides researchers an ES cell clone carrying the mutation of the gene of interest.

Because gene trapping involves the use of different mechanisms in generating mutations from the traditional knockout method, and its efficacy in targeting reproductive genes which often are expressed in later development or adult has not been fully established, it is necessary to examine the benefits and limitations of this technology, especially in the perspective of reproductive medicine so that reproductive researchers and physicians who are interested in mouse models could become familiar with this technology.

With this in mind, we provide an overview of the gene trapping mutagenesis method and its possible application to Reproductive Medicine. We evaluate gene trapping as a method in terms of its efficiency in comparison with traditional knockout methods and use an in-house software program to screen the IGTC database for existing cell lines with possible mutations in genes expressed in various reproductive tissues. Among over seven thousand genes highly expressed in human ovaries, almost half of them have existing gene trap lines.

Additionally, from 900 human seminal fluid proteins, 43% of them have gene trap hits in their mouse homologs. Our analysis suggests gene trapping is an effective mutagenesis method for identifying the genetic basis of reproductive diseases and many mutations for important reproductive genes are already present in the database. Given the rapid growth of the number of gene trap lines, the continuing evolution of gene trap vectors, and its easy accessibility to scientific communities, gene trapping could provide a fast and efficient way of generating mouse mutation(s) for any one particular gene of interest or multiple genes involved in a pathway at the same time. Consequently, we recommend gene trapping to be considered in the planning of mouse modeling of human reproductive disease and the IGTC be the first stop for people interested in searching for and generating mouse mutations of genes of interest.

Gene trapping is a high-throughput approach of generating mutations in murine ES cells through vectors that simultaneously disrupt and report the expression of the endogenous gene at the point of insertion. First-generation vectors trapped genes that were actively transcribed in undifferentiated ES cells. Depending on the areas in which they integrate, these vectors can be roughly divided into two classes:

  • promoter trap vectors and
  • gene trap vectors.

Promoter trap vectors contain promoterless reporter regions, usually bgeo (a fusion of neomycin phosphotransferase and b-galactosidase), and thus have to be integrated into an exon of a transcriptionally active locus in order for the cell to be selected for neomycin resistance or by LacZ staining. Gene trap vectors demonstrate more utility by their added ability to integrate into an intron. These vectors contain a splice acceptor (SA) site positioned at the 50-end of the reporter gene, allowing the vector to be spliced to the endogenous gene to form a fusion transcript. Later improvements include an internal ribosomal re-entry site (IRES) between the SA site and the reporter gene sequence; as a result, the reporter gene can be translated even when it is not fused to the trapped gene. Second-generation vectors have sought to trap genes that are transcriptionally silent in ES cells. Although these vectors still contain a promoterless reporter gene with a 50 SA sequence, the antibiotic resistance gene is under the control of a constitutive promoter. Consequently, antibiotic selection is independent from the expression of the trapped gene, whereas the expression of the reporter gene is still regulated by the endogenous promoter.

A disadvantage of these vectors is that all integration events give rise to resistant ES cells regardless of whether or not the vector has integrated into a gene locus. To increase trapping efficiency, a new class of polyA gene trap vectors was developed where the polyadenylation signal of the neo gene was replaced by a splice donor sequence, thereby requiring the vector to trap an endogenous polyA signal for expression of neo. These vectors were recently shown to have a bias toward insertion near the 30-end of a gene due to nonsense-mediated mRNA decay of the fusion transcript. An improved polyA trap vector, UPATrap, was developed to overcome this bias using an IRES sequence placed downstream of a marker containing a termination codon. Gene trap vectors are usually introduced by retroviral infection or electroporation of plasmid DNA, with each approach having its own advantages and disadvantages.

While relatively difficult to manipulate, retroviral gene traps display a preference toward insertion at the 50-end of genes, which is advantageous for generating null alleles. Moreover, the multiplicity of infection with retroviruses can be tightly controlled to a single trap event or simultaneous disruption in many genes. However, there may be a possible bias integration toward certain ‘hotspots’ of the genome.

In contrast, plasmid-based gene trap vectors integrate more randomly into the genome. This can, however, potentially result in a functional partial protein and a hypomorphic phenotype. Additionally, plasmid vectors usually result in multiple integrations in 20–50% of cell lines. The most common approach for identifying the gene trap integration site is to use 50 or 30 rapid amplification of cDNA ends (RACE) to amplify the fusion transcript. The sequence provides a DNA tag for the identification of the disrupted gene and can be used for genotypic screens. Mutagenesis screens can also be performed on the basis of gene function or expression, and data from an expression sequence combined with sequence tag information can elucidate novel expression patterns of known genes or to suggest gene function.

Gene trapping has proven to be an efficacious technique in mutagenesis compared with other methods such as

  • spontaneous mutations,
  • fortuitous transgene integration and
  • N-ethyl-N-nitrosurea (ENU) mutagenesis

We have been able to use our SpiderGene program to identify genes in reproductive tissues that are present in the IGTC database and moreover to narrow down those with restricted expression in the testis and ovary. Gene trapping possesses an enormous potential for researchers in the reproductive field seeking to create mouse models for a gene mutation. The improving versatility of gene trap vectors has enabled groups to trap an increasing number of genes in various organisms, including Arabidopsis, Zebra fish and Drosophila.

The gene trap effort has perhaps been the most extensive in the murine genome, with over 57000 cell lines representing more than 40% of the known genome. These large-scale screens will likely achieve the trapping of the entire mouse genome in the coming years, but the power of gene trapping will only be fully demonstrated by its usefulness in investigator-driven focused functional analyses.

In our laboratory, future work will focus on generating knockout mice in order to investigate gene function and to identify gene products that might have therapeutic value in reproduction. As screening efforts continue, gene trapping will continue to be a valuable tool in mouse genomics and will undoubtedly yield new discoveries in Reproductive Physiology and Pathology.

Source References:

http://www.ncbi.nlm.nih.gov/pubmed?term=Gene%20trap%20mutagenesis%3A%20a%20functional%20genomics%20approach%20towards%20reproductive%20research

 

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