Mitochondrial dysfunction and Correctible Reoccurring Aneuploid Conversion Syndrome (CRACS) A new category of treatable recurrent miscarriages and implantation failure common in Endometriosis and PCOS patients

Posted By Braverman IVF & Reproductive Immunology || 10-Sep-2015

We have previously posted blogs about our success with treatable recurrent miscarriage syndromes in:

- Women with 5 or more miscarriages (/Blog/2015/June/Patients-with-5-or-more-miscarriages-have-an-out.aspx)

- Women with history of euploid losses (/Blog/2015/August/Patients-with-at-least-one-loss-characterized-as.aspx)

We now discuss Correctible Reoccurring Aneuploid Conversion Syndrome (CRACS), a novel category of treatable recurrent miscarriage.

Recurrent pregnancy losses (RPL) have been mostly attributed to embryo chromosomal abnormalities, known as aneuploidy and as shown by recent studies (1-2) they can account for up to 80% of all losses. Up until now these have been considered random events and not treatable, with most being attributed to oocyte aging as one of the main factors involved in aneuploid embryonic losses (abnormal genetically).

However we, at Braverman Reproductive Immunology (BRI), now recognize that other conditions such as Endometriosis, PCOS, Diabetes, Obesity, Autoimmune Disease, as well as excessive IVF stimulations can also dramatically affect the oocyte quality. In particular, these conditions affect the mitochondria, one of the key organelles involved in oocyte quality and necessary for the correct division of the future embryo (you need normal mitochondria to supply the power for nuclear division, if mitochondria function is abnormal even a genetically tested normal embryo will fail to divide correctly and end up as an aneuploid embryo after transfer).
Diminished ovarian reserve (DOR) as evidenced by unexplained elevated FSH or low AMH in a patient under 40 is a reflection of this mitochondrial dysregulation, and yet left untreated by most IVF centers.

Mitochondrial dysfunction can be corrected by fixing the underlying conditions:

Endometriosis: excision therapy, supplements for mitochondrial optimization, immune modulation of follicular fluid (FF) during IVF or natural egg development.

PCOS: Metformin (Inositol), correct IVF stimulation protocols, immune modulation of FF.

Autoimmune Disease: immune modulation of FF.

IVF: correct stimulation protocols (low dose stimulations are better for this problem).

Obesity: Weight loss is important in those showing evidence of mitochondrial involvement (some patients see improved results after Gastric Bypass Surgery).

Mitochondrial replacement procedures: new therapies such as Augment to replace mitochondria for all of the above causes.

As we have seen and successfully treated this unique subset of patients, we have decided to regroup these recurrent aneuploid losses under the term:

Correctible Reoccurring Aneuploid Conversion Syndrome (CRACS)

In this blog, we will give you an overview on the causes inducing mitochondrial dysfunction, its impact on women fertility and the existing solutions to counteract its deleterious effects on oocyte/embryo quality.

I- Mitochondria

  1. What are the mitochondria?

Mitochondria are multitasking organelles present in the cytoplasm of a cell. They can be considered as “metabolic engine” whose main role is to produce ATP (energy) through a mechanism of oxidative phosphorylation. They also are involved in calcium signaling and apoptosis.

  1. What are their roles in oocyte and embryo development?

During the oocyte development (oogenesis), the egg accumulates a variety of substances such as proteins, RNAs, and organelles particularly non-replicating mitochondria that are crucial for early embryonic development (3). There are up to 250 000 copies of mitochondrial DNA which represents half of the total DNA content of the oocyte (4).

During meiosis, the oocyte goes from 46 chromosomes to 23 chromosomes to give rise to a mature (fertilizable) egg requiring energy that is provided by the mitochondria. After fertilization by a spermatozoid, mitochondria cluster around the zygote nucleus (which is the fusion of the oocyte and the spermatozoid, 46 chromosomes) to fuel cell division and are therefore vital to embryo division and development.
Mitochondrial DNA (MtDNA) does not replicate until after embryonic implantation at the blastocyst stage (5), therefore the fertilizable oocyte must have a sufficient amount of MtDNA to support embryo development up to this stage (6).

II- Mitochondrial dysfunction

Mitochondria has been demonstrated to regulate oocyte quality (7) and embryo potential for implantation through ATP production (8). Indeed, oocytes successfully fertilized in vitro, have higher copies of MtDNA than oocytes failing to fertilize (9) which translates into higher ATP levels (10).
During the follicular growth, the oocyte is particularly sensitive to factors present in the follicular environment (follicular fluid). Stress factors or inflammation triggered by diverse conditions (many of which were listed above) have a direct detrimental effect on mitochondrial function.

1. Causes of mitochondrial dysfunction

Obesity is a condition associated with ovulatory disorders, infertility, miscarriages and pregnancy complications. Indeed, obese patients have lower pregnancy success when using assisted reproductive technologies (ART).
However, these results are only seen with obese patients using own egg versus donor egg which clearly highlight that an “obese” environment can alter oocyte quality.
The poor oocyte quality reported in these patients could be the results of a mitochondrial dysfunction. In a mouse model, a high fat diet leads to:

  • Abnormal mitochondrial morphology undergoing apoptosis
  • Abnormal distribution of the mitochondria
  • Increased levels of ROS, marker of mitochondria damage
  • Abnormal oocyte metabolism
  • Abnormal spindles and chromosome alignment that lead to aneuploidy

As a result, the oocyte quality is severely affected and the embryo development highly compromised (11), leading to the possibility of aneuploid embryos.

Polycystic Ovarian Syndrome (PCOS)
PCOS has been associated with metabolic disturbances including the impairment of the insulin and glucose metabolism in the ovarian follicle. This can be a difficult diagnose to make as very few cases are “textbook” and therefore these patients are very often misdiagnosed or the diagnosis of any problem is missed entirely.
In women with PCOS, most likely secondary to abnormal insulin or glucose levels, a reduced expression of factors involved in the oxidative phosphorylation (mitochondrial function) has been reported (12-13). Moreover, an altered expression of key genes involved in the chromosomes alignment and segregation has also been reported (14) which can ultimately lead to aneuploidy. Lastly as many of these patients present to us simply with a history of hyperstimulation with IVF, the stimulation protocol alone in these patients, if untreated, can lead to mitochondrial damage (15). Altogether, these data showed that PCOS predisposes to chromosomal errors leading to a reduced oocyte quality and an increased rate of aneuploidy.

There are multiple causes of infertility in patients with endometriosis such as anatomic distortion, diminished ovarian reserve, and even peritoneal inflammation that can disrupt the endometrial receptivity (16). An accumulation of evidence tends to show that endometriosis alters the oocyte/embryo quality (17), most likely secondary to mitochondrial damage and whose sole presenting symptom to our practice many times is low AMH or elevated FSH that is unexplained. These patients typically have a history of aneuploidy either by PGD or testing of Products of Conception after a miscarriage.
A meta-analysis regrouping 22 IVF centers showed a significantly lower fertilization, implantation and pregnancy rates in patient with endometriosis in comparison with control subjects (18). Many recent studies have shown a link between endometriosis development and mitochondrial dysfunction with cell growth/apoptosis, oxidative phosphorylation, ROS production (all markers of mitochondrial function) being alter in endometriotic lesions.
In a recent study comparing N=50 women with endometriosis to N=39 healthy controls, a lower level of ATP production in cumulus cells (cells surrounding and supporting the oocyte’s growth and development) of women with endometriosis has been detected, which suggest an alteration of the nurturing role of these cells towards the oocyte (19). Furthermore, studies in mice showed that oocytes in the endometriosis group have abnormal meiosis with abnormal spindle and chromosome misalignment (20). More interestingly, when healthy oocyte are cultured in presence of peritoneal fluid from mice with endometriosis, the same oocyte alterations are observed and have been directly linked to mitochondrial dysfunction (21).
This strongly suggests that compromised peritoneal fluid with high levels of inflammatory factors and ROS could directly impact oocyte quality by altering mitochondrial function and disrupting the meiosis process. Excision of endometriosis implants can diminish these inflammatory factors and lead to improvement in embryo quality and reduction in aneuploidy.

2. Consequences of mitochondrial dysfunction on oocyte reserve (numbers of follicles available)

Diminished ovarian reserve (DOR) is defined by a reduced number of ovarian follicles and is characterized by lower AMH and elevated FSH levels as well as lower number of antral follicles.
By regulating the apoptosis process (self-destruction of cells), mitochondria play a key role in follicle atresia (loss of follicles) and are directly linked to DOR (22). Besides a diminished number of oocytes, DOR patients also show a decrease in oocyte quality with a higher number of oocyte aneuploidies, poor embryo development, and higher rates of pregnancy losses (23). This has been correlated with reduced oocyte MtDNA (24).


Although, the number of aneuploid losses decreases with the number of losses in RPL patients (i.e. after many losses an increasing number of those losses are euploid), the rate of aneuploid embryos is still higher than 65% (25).Therefore, we still need to address this group where age alone does not explain the recurrent aneuploid losses.

We have decided to regroup under the term “Correctible Reoccurring Aneuploid Conversion Syndrome or CRACS”, all patients suffering from recurrent aneuploid loss or implantation failure with an alteration in the oocyte quality that can be corrected.

IV- How to test for mitochondrial dysfunction in CRACS patients?

Besides the obvious evidence of poor embryo development after IVF, the following are also useful in determining possible mitochondrial damage.

1. Test: MitoGrade by Reprogenetics

Because mitochondrial insufficiency has been involved in chromosome segregation defects, in fertilization failure and in abnormal embryonic development, an evaluation of the mitochondrial DNA content in trophectoderm biopsies (embryos) could help select embryos that have the best potential to implant and lead to a viable pregnancy.
A recent study that analyses embryos MtDNA quantities (26) showed that:

  • MtDNA content is higher in embryos from older women (p=0.003)
  • MtDNA content is elevated in aneuploid embryos (p=0.025) regardless of maternal age
  • Implanted euploid blastocyst have a lower MtDNA content that those failing to implant (p=0.007)

The authors were able to determine a threshold above which implantation was never observed. This value was confirmed in an independent blinded prospective study.
The high level of MtDNA associated with a bad embryo quality/potential could be explained by the fact that lower quality embryo (due to the presence of defective mitochondria) are under stress and compensate by producing more energy as early as MtDNA replication resumes (at the blastocyst stage).

2. Follicular Fluid testing at time of IVF egg retrieval

We perform this routinely at BRI and are using these levels to diagnose abnormal follicular environments and recommend treatment strategies to improve oocyte development and reduce the risk of aneuploidy. This is in the early stages of development at our center (27).

V- Treatments

1. Surgery: laparoscopic excision of endometriotic lesions
Endometriosis is an inflammatory disease that create a noxious environment in the whole peritoneal cavity. Several studies have shown that oocyte quality is severely altered by endometriomas (lesions localized on the ovary) or more generally by endometriotic lesions regardless of their localization (16). The benefits of laparoscopy in endometriosis-related infertility cases have been shown in moderate (28) and stage 3 endometriosis subjects (29) where higher pregnancy rate have been reported post-surgery.

2. Supplements
Supplements have been shown to improve mitochondrial function (30). At BRI we have been recommending these supplements for years with great success and will soon be introducing our own supplement specifically for treating mitochondrial dysfunction in endometriosis.

  1. Augment treatment: Autologous Mitochondrial Treatment by Ovascience

This treatment is based on the facts that the transfer of isolated mitochondria in an oocyte can:

- increase ATP production

- prevent apoptosis

- promote embryonic development

It was long believed that women have a definite stock of oocytes (eggs) at birth that diminishes as menstrual cycles occur. A recent breakthrough discovery has shown the existence of quiescent egg cell precursors present in adult ovaries that can potentially develop into mature oocyte and give rise to fertilizable egg (31).
The Augment treatment consists of generating mitochondria in vitro from these egg cell precursors obtained after an ovarian biopsy. Mitochondria are then added into your egg during an IVF procedure which will help to “fuel” the high energy-demanding process of fertilization and blastocyst formation.

  1. Metformin use for PCOS patients

Metformin is a drug of choice to counteract the effect of PCOS on fertility. A prospective, randomized, double-blind, multi-center study (32) showed that the use of metformin for 12 weeks before and during an IVF cycle, significantly increases pregnancy (50% versus 33%) and live birth rates (48.6% versus 32% in controls).


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