Testing the Effects of Dehydroepiandrosterone on Follicular Maturation in the Ovaries
Packer Collegiate Institute, Brooklyn
Mentors: Olubanke Agunloye, Dr. Daylon James, Dr. Limor Man, Dr. Lindsay Kelly
Weill Cornell Endocrinology Laboratory
With the advancement of medicine over the years, there has been a large increase in cancer survivors. The current treatment for those with fertility issues is ovarian stimulation (OS) and later In Vitro Fertilization (IVF) through the use of synthetic hormones. OS increases the number of follicles that make it to the antral phase, allowing multiple antral follicles to be removed from the ovaries for IVF and later placed back into the uterus for implantation. However, ovarian stimulation is not a viable option for those with lower ovarian reserve because not many follicles move past the primordial phase at a time, making the antral follicle count (AFC) very low. Our lab looked for a new approach that could either increase the rate of growth of the follicles or decrease the percentage of death prior to the antral phase. After conducting research, we found other labs that perform similar experiments that used dehydroepiandrosterone (DHEA), a precursor to progesterone, estrogen, and testosterone, to solve this issue of infertility, but the results of these several studies yielded contradictory results. Our analysis of this hormone is currently ongoing and will be completed in a timely fashion, however, preliminary results of the sheer size of follicles that received DHEA leads us to believe that our hypothesis will be supported.
A common misconception in the biology of the ovaries is that it takes one month for ovarian follicles to mature and release an egg for ovulation. This, however, is not the case. Before the follicles reach the ovulatory phase, there is a period of about six to nine months (figure 1) in which, depending on their ovarian reserve, up to 1,000 follicles begin the process of growth towards their ovulatory phase (Gougeon et. al, 1986). The follicles housing the eggs in the ovaries go through 4 main stages of growth: primordial, pre-antral, antral, and finally ovulatory, when the egg is naturally released and the menstruation process begins (Gougeon et. al, 1986). By the arrival of the antral phase, around 99% of the eggs that awoke from their dormant stages have died. This brings about a problem for those who want to go through the process of IVF and have their eggs removed. A low number of antral follicles for removal and later insemination decreases the chances of a live birth occurring as not every egg will implant properly (Mayo Clinic, 2018). To combat this, the patient is given synthetic hormones to stimulate their ovaries into growing more antral follicles than the naturally occurring 1%. The use of ovarian stimulation even without IVF in turn, increases the likelihood of a live birth as there are more eggs moving past the antral phase and into ovulation.
However, in patients with a naturally low ovarian reserve, even with ovarian hormone stimulation, there is a much lower antral follicle count and consequently, a low likelihood of a live birth naturally or through IVF. Low follicle count occurs naturally later in life as you age. However, young patients who had cancer as children are now attempting to have children at no avail for the same reason. This is due to the fact that chemotherapy drugs do not only attack cancer cells; they aim for any type of cell that replicates at a high rate. This is very helpful since tumors, the target of chemotherapy, replicate at an extremely rapid rate. However, other parts of the body, like the roots of one’s hair or ovaries, also have cells that replicate at similar rates. This causes some of the symptoms, like hair loss, that many people associate with chemotherapy. Before a patient reaches adolescence and begins puberty, ovarian stimulation cannot be performed, because the body is not ready for the introduction of hormones. Consequently, children undergoing chemotherapy are unable to have any of their eggs removed for future use, in turn rapidly depleting their ovarian reserve and even bringing upon early menopause post cancer treatment. Around 1980, the survival rate for children with cancer was around 1/10. This has now greatly increased to a survival rate of around 8/10 patients. Despite the cancer being a part of their past, with the influx of cancer survival, there is now a question of quality of life after the disease. This question pushed our lab to look for an alternative approach to better the survival of early-stage follicles. In this alternative approach, we aim to either decrease the percentage of follicular death from 99% to around 80%, which would make a huge difference in fertility, or increase the rate of growth of the follicles.
DHEA is considered a precursor steroid to progesterone, testosterone, and estrogen and is an active hormone in human physiology (Powrie and Smith et. al, 2018). This hormone is found within all humans but becomes less prevalent as humans age despite it being the most widespread steroid within the human body (Rutkowski and Sowa et. al, 2014). From numerous animal studies, low levels of the hormone have been associated with involuntary age-related changes (Rutkowski and Sowa et. al, 2014). It is currently being sold all over the market as an over the counter anti-aging panacea that improves physical and psychological well-being (Rutkowski and Sowa et. al, 2014). Experiments have been conducted on DHEA’s effect on follicle maturation but with varying conclusions about its efficacy (E E Baulieu,1996). Our experiment aimed to test whether or not this decrease in DHEA and increase in infertility as patients age is just a coincidence or if it possibly has some type of cause and effect relationship. If it is found that DHEA affects the follicle count in the ovaries, then it could be used as a medicinal treatment for infertility.
We believe that with a daily introduction of DHEA in mice, there will be more follicles in the antral phase compared to the control. The past experiments that this experiment has been based on only used models with and without DHEA supplementation. We decided to use those same models in addition to two models with 10 days with and 10 days without DHEA in hopes of seeing a midway point in between the results from the models with and without DHEA.
Figure 1: The chronology of normal folliculogenesis in human ovaries and depicting their size and looks over the six to nine month period.
An experimental design was created to outline the process that would take place prior to data collection (figure 2).
Mice and DHEA/Sesame Oil injections
4 groups of 3 immunodeficient mice (6 ovaries) each were given an injection of their body weight (g) * 5µl of either DHEA (active agent) or sesame oil (control) over a span of 19 days (around 12:00 PM each day). Sesame oil was chosen as the control because it has been used in previous experiments at our lab so its lack of effects are known and the mice enjoy the taste on their fur. At the end of the experiment had their ovaries extracted to observe follicular development under four conditions; Condition 1: DHEA injected into the ovaries of the mice over the entire 19 day period. Condition 2: Sesame oil injected over the entire 19 day period. Condition 3: Sesame oil injected over the 10 day period. Following the first 10 day period, DHEA was injected into the ovaries. Condition 4: DHEA injected over the 10 day period. Following the first 10 day period, sesame oil was injected into the ovaries.
On day 19, all the mice were killed and their ovaries were extracted. After removal, ovaries went through tissue preparation for histological slide production for future observation. The fat and follicular dust were removed. The ovaries were then weighed individually on an Adventurer Pro to see if any preliminary conclusions could be made between the ovaries of the mice.
Then the tissues were fixated in a 4% formaldehyde solution for 24 hours (R&D Systems). Ovaries were then equilibrated in 30% sucrose solution to prevent tissue damage from the expansion of water crystals during freezing (Parry, 2018). Then they were quick frozen in liquid nitrogen to further prevent the formation of water crystals and to prep for sectioning. The tissues were then sliced into 10 µm thick wedges for proper slide fixation. The tissue then went through a process of haematoxylin/eosin histological staining (H&E) which gave the tissue blue, violet, and red hues for observation under the microscope.
Figure 2: Experimental design with basic expectations written down as a guide for data collection. This was written out as a thought process with notes to aid in understanding. The bottom right side illustrates the expectation of an increase in AF. Directly above is the method of comparing the percentages of the number of follicles in each follicular phase between the 4 experimental groups. In the middle, above the hypothesis, is our own scaled version of Alain Gougeon’s model of folliculogenesis.
We are currently observings and conducting AFCs under a microscope for each of the four experimental groups. Data is being written down on sheets of paper to later be transferred to an online spreadsheet for increased ease of comparison. We are also recording the number of follicles in stages prior to the antral phase to get a better comparison between the experimental groups and control. Once we are finished collecting data for the experiment, we will begin to analyze the differences between the mice who were given supplements of DHEA and the mice who were just given sesame oil, as a control. Currently, we have preliminary results based off of eying scaled samples of mice ovaries with an without DHEA (figure 3). No conclusion can be made off of this alone, but from what we can see, the ovarian tissue of the supplemented mouse is larger than the one of the control mouse which leads us to believe that our hypothesis will be supported once the AFCs are completed.
Figure 3: Side by side sectionings of the ovaries of a mouse injected with oil for 19 days (left) and a mouse injected with DHEA for 19 days (right).
If our hypothesis is supported by our data as we predict with our preliminary observations, this will mean that those living with low follicle count, be it from chemotherapy, old age, or other factors will be one step closer to living with the option of having kids. There are currently other alternatives being explored like the freezing of ovarian tissue at a young age or before chemotherapy to later be implanted back into the patient (Meirow and Ra’anani, 2016). This however is not a viable option for all types of aggressive cancers in which the chemotherapy can’t be delayed. The reimplantation of tissue already affected by chemo, despite its positive results, has not been extensively researched to identify any real risks. One drawback of this experiment is the use of mice. Humans and mice have very similar reproductive anatomy, but one difference is the timing of the maturation cycle. Mice’s follicles mature through its phases in about 20 days while for humans it takes about 10 times that (Gougeon et. al, 1986). We injected the mice with DHEA for 19 days which is almost their complete maturation cycle. If we want to bring this research into the human field, it would not be practical to inject a human with DHEA for 6-8 months daily. More tests will have to be done to shorten the administration period while retaining the foreseen benefits.
I would like to thank Dr. Daylon James for accepting me as an intern at his lab and mentoring me in this research project. I would also like to thank Dr. Limor Man and Dr. Lindsey Kelly for working very closely with me on the data collection and teaching me the tools for this process. Lastly, I would like to thank Mr. Holzinger for guiding me in this program for the last two years as well as the other independent science research coordinators at my school.
Gougeon, Alain. (1986). Dynamics of Follicular Growth in the Human: A Model from Preliminary results. Human reproduction (Oxford, England). 1. 81-7. 10.1093/oxfordjournals.humrep.a136365.
E E Baulieu; Dehydroepiandrosterone (DHEA): a fountain of youth?, The Journal of Clinical Endocrinology & Metabolism, Volume 81, Issue 9, 1 September 1996, Pages 3147–3151, https://doi.org/10.1210/jcem.81.9.8784058
Meirow, Dror and Ra’anani, Hila (2016). Transplantation of frozen-thawed ovarian tissue demonstrate high reproductive performance and the need to revise restrictive criteria. American Society for Reproductive Medicine, Vol. 106, No. 2, August 2016.
Nicola Parry (2018). How Histology Slides are Prepared. (2017, January 16). Retrieved from https://bitesizebio.com/13398/how-histology-slides-are-prepared/
Rutkowski, K., Sowa, P., Rutkowska-Talipska, J. et al. Drugs (2014) 74: 1195. https://doi.org/10.1007/s40265-014-0259-8
Powrie and Smith et. al. (2018) Central intracrine DHEA synthesis in ageing-related neuroinflammation and neurodegeneration: therapeutic potential, The Journal of Neuroinflamation, https://www.ncbi.nlm.nih.gov/pubmed/30326923
In vitro fertilization (IVF). (2018, March 22). Retrieved from https://www.mayoclinic.org/tests-procedures/in-vitro-fertilization/about/pac-20384716
Ovarian hyperstimulation syndrome. (2017, August 03). Retrieved from https://www.mayoclinic.org/diseases-conditions/ovarian-hyperstimulation-syndrome-ohss/symptoms-causes/syc-20354697
Protocol for Making a 4% Formaldehyde Solution in PBS. (n.d.). Retrieved from https://www.rndsystems.com/resources/protocols/protocol-making-4-formaldehyde-solution-pbs