Can metformin prevent embryo aneuploidy?

A dividing cell - microtubules in 'green' and chromosomes in 'red'

Metformin is an anti-diabetic drug used to treat type II diabetes alongside diet and exercise. It functions by increasing the sensitivity of our body’s cells to insulin (decreases insulin resistance) and thus by helps in the proper utilization of glucose.  It has no direct effect on insulin secretion from pancreas and so it does not alter the insulin level in our body. Hence the risk of dangerous hypoglycemic episodes is very low when compared to other anti-diabetic drugs. Metformin has a very high safety record and is used in clinical practice for more than 50 years. 

Metformin is now used widely used for treating Polycystic Ovarian Disease (PCOD). Insulin resistance and obesity are common among women with PCOD. They have high levels of androgens in their body. They suffer from absence of ovulation and hence lack regular menstrual cycles too. Metformin treatment of PCOD women decreased circulating insulin levels, corrected hyperandrogenism (presence of high level of androgens) and helped in the resumption of regular ovulation. As a result many PCOD women are able to conceive with the help of metformin. Continuation of metformin during the first trimester reduced miscarriage rates in women having PCOD (women with PCOD are prone to miscarriages) and continuation of metformin throughout pregnancy prevented gestational diabetes.  PCOD is a multifaceted disease and the exact mode of metformin action in helping PCOD patients is still unknown. Another interesting information regarding metformin is, it is touted as a gerosuppressant (anti-aging drug) and it prevented reproductive aging too (estrous cycle in mouse treated with metformin remained regular even in older age while in control animals estrous cycle became irregular with age!) (PMID: 18728386).  Metformin works as a calorie restriction mimetic.

Several studies have been carried out to find whether metformin treatment of women with and without PCOD increases the implantation rate and pregnancy rate during IVF treatment. Some studies found that metformin increased egg quality, pregnancy rate and implantation rate in women with PCOD and some failed to show any benefit. But metformin treatment of women with PCOD during IVF was found to prevent ovarian hyperstimulationsyndrome. A multi-centre, prospective, randomized, double-blind study conducted in 2011 showed that metformin treatment of non-obese PCOD women undergoing ART cycles improved pregnancy rate and live birth rate but the clinical pregnancy rate remained the same between the metformin treated and the placebo group.

The exact mode of action of metformin in preventing miscarriages, improving pregnancy and live birth rates remains unknown. It is hypothesized that decreased insulin and androgen levels contribute to this beneficial effect. It is known that more that 60% of miscarriages happen because of embryo aneuploidy (a form of genetic aberration leading to abnormal chromosome number in the embryo). So the question arises whether metformin does something to prevent embryo aneuploidy. To answer this question I tried to look into the signaling pathway activated by metformin and its effect on cell division. 

 Metformin activates a signaling pathway called AMP Kinase. AMP Kinase is called the energy sensor of the cell. When the energy level is low within the cell AMP Kinase senses this energy deficit and switches on activities within the cell which produces more energy and switches off activities which consumes energy. AMP Kinase also functions as a tumour suppressor and metformin which activates AMP Kinase has been proved to posses anti-cancer properties. Interestingly it was found that, AMP Kinase is necessary for proper cell division in drosophila and lack of functional AMP Kinase subunits lead to abnormal chromosome segregation during mitosis (which is the reason for chromosomal abnormalities or aneuploidy) and increased polyploid cells. It was also found that AMP kinase regulates the stability of spindlemicrotubules, the structures responsible for proper chromosomal segregation during cell division. Another publication stated that AMP Kinase activators like metformin can selectively induce apoptosis in aneuploid cells. 

A couple of studies were also done by adding metformin to the culture medium in which embryos of experimental animals were grown. They observed increased blastocyst formation when metformin is present in the culture medium containing insulin (PMID: 16107611). When AMP Kinase was activated using metformin in mouse embryos there was decreased apoptosis and increased pregnancy rates (PMID:17575082).

Can the beneficial effect of metformin (decreased miscarriage rates) in human reproduction be due to the result of metformin’s ability to prevent cell division errors by activating AMP Kinase? Isn’t aneuploidy the major reason for miscarriages? Are laboratory culture conditions (like excess nutrients in the culture medium, over activation of IGF-1 pathway as a result and complete supression of AMP Kinase pathway) a culprit for producing lot of aneuploid embryos? Can adding metformin to embryo culture medium prevent aneuploidies arising due to mitosis? Does metformin uptake by women improve reproductive outcome by reducing aneuploidy in their oocytes? There are many unanswered questions and perhaps research in this area will help in finding the plausible connection between metformin use and embryo anueploidy prevention!

Comprehensive Chromosome Screening (CCS)-panacea or pipe dream? - Part II

You can read the first part of this series here.

Karyotype showing aneuploidy of chromosome 21

 Embryo aneuploidy

An aneuploid embryo contains abnormal number of chromosomes in their cells. A human embryo should contain 23 pairs of chromosomes (46 chromosomes) in every cell. Chromosomes carry the genes. Genes encode the information for producing proteins. So when abnormal number (more or less)  of chromosomes are present in a cell more or less than the required amounts of proteins are produced. This can either kill the embryo by arresting its development (embryo doesn’t implant or even if it implants the pregnancy gets terminated spontaneously!) or can cause severe abnormalities in babies which are born out of an aneuploid embryo. For example a baby with Down’s syndrome carries three copies of 21^st chromosome (an entire extra chromosome or part of it!)  in its cells.

Aneuploid embryos can arise as the result of:

1) Errors in cell division during oocyte (egg) development.
2) Errors in cell division during sperm development.
3) Errors in cell division during an embryo development.

90% of embryo aneuploidy are due to cell division errors in oocytes and only 10% of the errors are contributed by the problems arising during sperm development. As a woman age the number of eggs with abnormal chromosome number increases.

Eggs and sperms are formed by a special type of cell division called meiosis.  The reason why we are so different from our parents and ancestors (in physical and mental traits) is because of genetic recombinations taking place during meiosis. Meiosis introduces genetic variations in offspring. Meiotic division is reponsible for the production of eggs and sperms. Eggs and sperms carry only half the number of chromosome (23 unpaired chromosomes) and during fertilization the full chromosome complement (46 chromosomes) is restored. When a chromosomally abnormal egg is fertilized by a normal sperm or vice versa (or if a chromosomally abnormal egg is fertilized by a chromosomally abnormal sperm) the resulting embryo will be genetically defective or aneuploid. When chromosomal errors occur during meiosis it affects the entire embryo. All the cells within the embryo will be aneuploid. Aneuploidy of meiotic origin is almost always lethal to the embryo or to the fetus. But approximately one third of aneuploid oocytes derived from sequential errors in the first and second meiotic divisions resulted in a balanced karyotype, representing a possible phenomenon of “aneuploidy rescue”

An embryo can also acquire genetic errors during its cell division. The embryo divides by a type of cell division called mitosis. The first three cell division of an embryo is extremely prone to genetic errors. An embryo activates its own genome when it reaches the 8-cell stage. Until then the embryo depends on maternally derived gene transcripts and proteins stored in the oocyte. For the prevention of aneuploidy formation high levels of mitotic and cell cycle proteins are necessary. The quality of the stored gene transcripts and protein could diminish over time by the accumulation of radiation or toxic agents, oxidative stress, compromised mitochondria or telomere shortening. This can lead to a defective cell cycle checkpoint mechanisms (especially in women of advanced maternal age), which may lead to chromosomal segregation errors in the first few cell division of human preimplantation embryos. When a zygote (embryo in 2pn stage) divides it gives rise to a two celled embryo. When genetic error occur in the first division then the entire embryo will be aneuploid. When a two celled embryo divides one cell division can be normal and the other defective. If such an error occurs one-half of the embryo will carry cells which will contain chromosomal abnormality and the other half of the cells will have a normal genetic make-up. When one-quarter of the cells in an embryo are chromosomally abnormal then it means that the chromosomal aggregation error occured in the third division. An embryo which carries both genetically normal (euploid cells) and geneticall abnormal cells (aneuploid cells) is said to be mosaic and the phenomenon which leads to the formation of mosaic embryos is called mosaicism.

Can mosaic embryos develop into normal babies?

Single cell comparative genomic hybridization analyses of normal IVF embryos showed that 75% of all the IVF embryos were mosaic. Out of these embryos 55% were diploid-aneuploid mosaic and 55% of all blastomeres from these embryos were diploid (PMID: 21531753). Mosaicism is found to be more in blastocysts when compared to cleavage stage embryos. This implies that mitotic errors could have occurred in later cell divisions and not necessarily in the first three mitotic division of a zygote. High rate of diploid-aneuploid mosaicism in blastocysts also implies that mosaic embryos more easily reach blastocyst stage as compared to embryos containing only aneuploid blastomeres. Do these mosaic embryos develop into normal babies? An experiment conducted in mice showed that only 20% of euploid cells in the blastocyst are enough to give rise to a normal mouse. This shows that blastocysts might have inherent mechanisms to correct their genetic defects. There are many speculations regarding such mechanisms. It is assumed that the blastocyst rearrange their blastomeres in such a way that the aneuploid cells are pushed towards the periphery forming the trophectoderm leaving only the diploid or euploid cells in the inner cell mass. This could explain the phenomenon of confined placental mosaicism. The other school of thought is abnormal cells are removed from the blastocysts via apoptosis and only the euploid cells survive. This leads to the accumulation of normal cells in the embryo.

It was also shown that frozen-thawed human embryos that lost nearly half of their blastomeres are still able to result in live births. This clearly shows that not all blastomeres of human pre-implantation embryos are necessary for developing into a full-fledged baby. Transfer of two tetraploid blastocysts (as identified by trophectoderm biopsy) in a woman has resulted in the birth of a normal male infant (PMID: 19608167). Embryonic stem cell lines developed from aneuploid embryos were found to have normal chromosomal make-up.These evidences clearly show that mosaic embryos have the potential to ‘self-correct’ and develop into normal babies. Might be, the type of mosaicism, the percentage of mosaic cells in an embryo and the inherent ability of the embryo to correct itself, determines whether a mosaic embryo could develop into a baby or not.

Further researches are needed at this point to determine whether embryo mosaicism is actually a pathological or physiological mechanism.

 

Array Comparative Genome Hybridisation

Fluorescence in situ hybridization (FISH) used to be technique for screening genetic abnormalities in pre-implantation embryos. However, this was unable to screen all the chromosomes in an embryo for genetic abnormality ; and was  also highly labor intensive. This has led to the development of advanced cytogenetic techniques which can scan all the 23 pair of chromosomes for genetic errors. One such technique is called array comparative genome hybridization (aCGH). The primary advantage of CGH is its ability to detect aneuploidies, deletions, duplications and/or amplifications of any locus represented in an array. One assay using this technique is equivalent to thousands of FISH experiments. Array-CGH has been successfully used to detect submicroscopic chromosomal aberrations which are also called as copy number variants (CNV).

Advantages of aCGH

1) Useful for comprehensive chromosome screening (CCS).

2) It has very high resolution (can detect submicroscopic variations in genome).

3) Quicker results and it is not labor intensive, since it is automated

Limitations of aCGH 
 
1) Whole genome screening using aCGH can generate data that may be difficult to interpret.

2) It can detect even minute alterations in genome which might have no established clinical relevance.

3) Clinical confidence of aCGH is still in question. Many researchers advocate FISH confirmation of the results obtained using aCGH.

You can read the next part here.

Comprehensive Chromosome Screening - panacea or pipe dream? - Part-1



Chromosomal screening of human blastocyst using CGH

I have had 19 embryos transferred  to my uteurs. Only one of it implanted but it failed to develop into a healthy infant. This shows that not all embryos produced via IVF are able to develop into a much desired baby. Many women who have undergone IVF ask this question frequently 'why didn't my embryo implant?' Although there is no easy answer for this question scientists are trying hard to decipher this puzzle. Why don't all embryos develop into a baby? What can be done to improve the success rate of IVF? Can we achieve 100% success rate in ART? Is there a way to determine which embryo will develop into a healthy baby? A very recent technological advancement which appears to be promising in improving IVF success rate is Comprehensive Chromosome Screening (CCS). It utilizes modern genetic techniques to find out embryos which are genetically normal. Will such modern genetic screening techniques take IVF to new heights? Will the success rate of IVF improve dramatically in the coming years? These are all very interesting questions and I have a made a review which might answer some of the above questions atleast partly. My next couple of posts will talk about embryo aneuploidy, aneuploidy detection techniques and whether it will really make a difference in the field of ART. This topic might be a bit more scientific and difficult to understand. Please write to me if you want to clarify any doubts.



Why do we need to screen embryos for genetic defects?

Not all the embryos which enter the uterine cavity will implant and develop into a baby. If we are able to pinpoint which embryo has the potential to develop into healthy infant then the success rate of an IVFcycle will greatly improve. This will also pave way for elective single embryo transfer (e-SET) which in turn will prevent the dangers associated with multiple gestations. The universally accepted truth in reproductive biology is, younger the women; greater is her ability to conceive and carry a baby to term. As a women age the quantity and quality (genetic quality!) of her eggs decline. As a result it becomes difficult for older women to conceive and even if she conceives many pregnancies are lost in the earlier stages of gestation. They are also prone to giving birth to babies with genetic abnormalities. The genetic analysis of fetal remains from spontaneous abortion samples (from younger and older women) revealed that more than 60% of fetuses stopped developing because of the presence of chromosomal aberrations (incorrect chomosomal number or content in the cells). Aneuploidy is the most common genetic aberration present. Oocytes from older woman are more prone to develop chromosomal aneuploidies. This knowledge and the failure of most of the embryos generated via Artificial Reproductive Technology (ART) to develop into babies (only 19% of transferred embryos were delivered) lead to the genetic screening of embryos for chromosomal defects. The field which deals with the screening of embryos for their aneuploidy status is called preimplantation genetic diagnosis (PGD). Until recently, the widely used screening technique in the field of PGD is called Fluorescence in situ hybridization (FISH). Preimplantation genetic screening (PGS) using FISH failed to show improvement in the implantation rates and delivery rate for women of advanced maternal age. This lack of improvement in pregnancy rate was attributed to the limitation of FISH technique itself. Using FISH it is only possible to screen a very limited amount of chromosomes (5-12 chromosomes) for genetic abnormality or aneuploidy. An anueploidy can strike any of the 24 different chromosomes present in a human embryo. It is argued that FISH failed to detect embryo aneuploidy in many of the embryos and as a result there is no improvement in pregnancy rate of women of advanced maternal age even with PGS. Recently limitations in FISH technique are overcome by the introduction of genetic screening techniques which could screen all 23 pairs of chromosomes in human embryos. This screening technique is called Comprehensive Chromosome Screening (CCS) and it employs genetic screening techniques like whole genome amplification combined with qPCR,SNP microarray-based analysis or array-comparitive genomic hybridization (aCGH).

More than 50% of embryos generated via IVF are found to carry some forms of aneuploidy. ( This is true of embryos created in the bedroom as well ! Human reproduction is remarkably inefficient). Even embryos which appear perfectly normal under the microscope (scored using cell size and number, presence of multinucleation, percentage of fragmentation and cleavage rate) are found to carry genetic abnormalities. Surprisingly, an ugly looking embryo under the microscope can be genetically normal and a beautiful looking embryo can be genetically abnormal. The high aneuploidy rates found in embryos might explain the low implantation rate and birth rate per embryo transfer. Most clinics around the world have a success rate of 40-45% per IVF cycle. This rate goes down drastically for women over 38 years of age. Advanced maternal age also leads to high level of spontaneous abortion because of the implantation of genetically defective embryos which fail to progress normally in utero .The embryo chromosomal abnormality rate is about 40% for women aged up to 29 ; and it increases up to 80% for those aged 40 or above. This increase in genetically defective embryos in older woman is usually due to the increase in trisomies. More than 50% of aneuploid embryos have the capacity to develop and implant. Some of these embryos can even develop into babies , which are genetically abnormal. Aneuploidies that can result in viable pregnancies include chromosome 13, 18, 21, X and Y. The common genetic anomalies present in embryos are:

1)      Trisomy (62%)

2)      Triploidy (12.4%)

3)      Monosomy X (10.5%)

4)      Tetraploidy (9.2%)

5)      Structural anomalies (4.7%)

 

Transferring euploid (chromosomally normal) embryos might result in:

1) Increased implantation rates and live birth rate per embryo transfer and hence encourage the use of elective single embryo transfer (e-SET). This in turn will prevent multiple pregnancies and the risks associated with this.

2) Decreased incidence of spontaneous abortions (60%-70% reduction in spontaneous abortion rate)

3) Reduced risk of carrying and delivering a genetically abnormal baby.

Who might benefit from embryo screening?

1) Patients with inherited genetic disorders.

2) Patients who suffer recurrent pregnancy loss (RPL) because of advanced maternal age or from rare chromosomal translocations.

3) Women of advanced maternal age (AMA) who are at increased risk for carrying a genetically abnormal child.

4) Patients with recurrent implantation failure (RIF) or repeated IVF failure. Patients who have had more than 10 good looking embryos transferred into their uterus without ever achieving a pregnancy are considered as having recurrent implantation failure.

5) For patients who suffer from unexplained infertility. Genetic screening of embryos might help in understanding whether unexplained infertility is embryo related or implantation related. 

You can read the next part here.