Jan Friberg M.D., Ph.D.
Inflammation and oxidative stress are intimately involved in the creation of polycystic ovarian syndrome (PCOS). The initial events that leads to the formation of PCOS are unknown. Inflammation with production of cytokines such as Tumor Necrosis Factor alpha. Interferone gamma, Interleukin 1beta and interleukin 6 stimulate androgens production that create abnormal folliculogenesis with excessive number of small follicles, death of follicle (follicular apoptosis) and abnormal vessel formulation in the ovary. The follicles that develop have therefore very often abnormal oocytes that do not easily fertilize and develop. Many techniques have been used to treat these patients infertility but in a proportion the attempts are not successful.
Mesenchymal stem cells (MSC) are known to be anti-inflammatory, anti-apoptotic, immunomodulating, and create an antifibrotic environment. MSC have recently been applied in human medicine with great success since they are easy to handle and quite safe and well tolerated. In infertility treatment they recently have been used to treat premature ovarian failure and low egg production in IVF procedures. MSC have now also been introduced for PCOS treatment and experimentation indicated that they offer a great promise to correct the PCOS pathology.
Polycystic ovarian syndrome (PCOS) is the most common endocrine disorder affecting 8 – 10 % of all women of reproductive age (March et al 2010). Several diagnostic criteria for PCOS have been defined but the most commonly used is the one proposed at a meeting in Rotterdam in 2003, and includes ovulatory dysfunction, hyperandrogenism, and multicystic ovaries. PCOS is associated with insulin resistance, hyperinsulinemia, type 2 diabities, obesity, dyslipidemia, and increased risk of cardiovascular disease – conditions that seriously challenge womens long term health. In addition to the endocrine and metabolic changes that are common in PCOS, the disease complex also affects the uterus and puts PCOS women at risk for endometrial hyperplasia, menomethrrhagia and development of endometrial cancer. In spite of years of research, the etiology of PCOS is still unknown and no effective treatment is available (Moran et al 2010, Franks 1995, Giudice 2006).
General guidelines for treatment of PCOS is life-style changes with exercise and dieting intervention. Birth control pills are often used to control irregular cycles and metformin, inositol and other antihyperglycemic drugs are often needed to control hyperglycemia, insulin resistance, and type 2 diabetes. For infertility treatment of PCOS patients, Clomid and Letrozole, sometimes in combination, are first hand drugs to use but gonadotropin stimulation and IVF have a place in the treatment but the risk of hyperstimulation is always present. In the past, ovarian wedge resection and now laparoscopic or transvaginal ovarian drilling have become popular but an increased risk for adhesion formation with entanglement of the Falloppian tubes is relatively high and may create another fertility obstacle.
Low grade inflammation associated with hyperandrogenism, oxidative stress, and hyperinsulinemia have been linked to the development of PCOS (Gonzales 2012, Murri et al 2013, Escobar-Morreale et al 2011, Goodarzi et al 2011, Showell et al 2013, Rocha et al 2019).
In patients with PCOS, the ovarian tissue displays an aggressive infiltration of lymphocytes and macrophages (Bremer 2010, Rosenfield & Ehrmann 2016,). The inflammatory response stimulates androgen production that induces follicular atresia and ovarian dysfunction. Infertility becomes a prominent symptom in these patients and despite aggressive infertility therapy many patients remain infertile. On the laboratory side the inflammation and resulting oxidative stress manifest itself with elevated levels of c-reactive protein (CRP), tumor necrosis factor alpha (TNF-α), interferon gamma (IFN gamma), interleukin 1-β (IL-1 β), interleukin 6 (IL-6), interleukin 18 (IL-18), anti-nuclear antibodies (ANA) and many other autoantibodies (Rezvanfar et al 2016, Amato et al 2003, Yang et al 2011, Gao et al 2016). Thesee substances are all pro-inflamatory factors that can activate immune cells such as neutrophils and lymphocytes and create inflammation.
The immunological component with oxidative stress and inflammatory changes in PCOS have over the past few years come into focus as a target for new treatments of PCOS. Conditions similar to PCOS can be induced in animals with injection of for example testosterone enanate, dehydroepiandrosterone or letrozole. The injected animals develop a typical PCOS appearance with ovarian cysts, hyperandrogenemia, ovarian dysfunction, hyperinsulinemia, insulin resistance, dyslipidemia, type 2 diabetes changes and even hyperplastic changes in the uterus. Because of their anti-inflammatory , anti-oxidative, anti-apoptotic and immunomodulating properties, mesenchymal stem cells (MSC) have then been applied with the intension to reverse the PCOS changes. MSC have a long positive history in the treatment of many auto-immune and inflammatory diseases in many specialties (Zappia et al 2005, Jones & Mc Faggart 2008, Anzalone et al 2011). MSC can suppress immunophenomenon from inflammatory cells and elevated levels of pro-inflammatory cytokines enhance and augment the anti-inflammatory properties of MSC and also increase the “homing in” effect of MSC to an area in the body with a disturbed microenvironment (Chapel et al 2003, Caplan 2009,Xiao et al 2013, Mirabolghsemi and Kamyes 2017, Xiao et al 2019). MSC are also known for their ability to improve ovarian function in both animals and humans after gonadotoxic treatment (see review by Rosario & Anderson 2020, Chang et al 2021, Mawet et al 2021).
The anti-inflammatory and immunomodulatory properties of MSC stimulated Kalhori et al (2018) to evaluate if MSC treatment would show any benefits and improve symptoms in PCOS. They injected bone marrow derived MSC intravenously in induced PCOS mice and were able to document significant improvements in PCOS symptoms with increases in FSH levels and total antioxidant capacity and decreases in testosterone and LH levels with fewer apoptotic cells in the ovary. They concluded that MSC therapy could be of value in the treatment of PCOS. Park et al (2019) also evaluated the effect of MSC on the inflammatory response and blood vessel formation in mice with induced PCOS. They used direct intraovarian injection of the MSC and concluded that PCOS can be reversed by intraovarian injection of the MSC. This information was further extended by Xie et al (2019) who showed that administration of MSC significantly downregulated the expression of proinflammatory TNFα, IL-1, IFN-gamma, and fibrosis-regulated genes. Inflammatory M1 macrophages and B-cells were significantly reduced while M2 macrophages (wounded healing cells) and regulatory T-cells were increased after MSC treatment. The mechanism behind the improvement of PCOS symptoms after MSC treatment has been examined and both Bone Morphogenic Protein 2 (BMP-2) and Interleukin 10 (IL-10) have been singled out as important mediators of the ability of MSC to improve PCOS (Chugh et al 2021a, Chugh et al 2021b) but many more growth factors and cytokines are likely to be identified . In PCOS where the ovarian theca-cells proliferate abundantly (Rosenfiels & Ehrman, 2016, Bremer 2010) BMP-2 is able to inhibit this growth (Glister et al 2013)
Sophisticated research over the past few years has focused on mechanisms creating the immunosuppressive and anti-inflammatory effects of MSC. Cell to cell contact and the secretome with exosomes are strongly considered to be the main functioning factors (Yoon 2019, Fan et al 2020). Another important factor is the ability of MSC to migrate towards the damaged microenvironment (Chapel et al 2003, Caplan 2009, Ullah et al 2019). The capacity of MSC to turn themselves into the different supporting cells in the ovary is also a significant factor (Takehara et al 2013, Li et al 2019, Yoon 2019). miRNA (Micro RNA) present in exosomes from MSC also appears to be an important mediator of MSC effects and play a regulatory role in follicle development and oocyte maturation (Zhang et al 2020). Exosomes are membrane bound extra cellular vesicles that can be secreted from stem cells (Huang et al 2016, Ding et al 2020). Non-coding miRNA from MSC exosomes have also emerged as important mediators of ovarian rejuvenation effects after stem cell treatment. miRNA regulate gene expression in MSC based therapy (Ali et al 2020, Yang et al 2020). Several different miRNA;s have been shown to support granulosa cell proliferation and inhibit cell atresia by targeting the anti B-cell lymphoma 2 like protein II (a protein that promotes cell death) (Fu et al 2008, Xiao et al 2016, Feng et al 2019, Li et al 2019, Sun 2019). In PCOS for example miRNA-323-3p in exosomes is downregulated with an increase in FSH, LH and testosterone while estradiol levels drop resulting in cell dysfunction and apoptosis in oocytes and cumulus cells (Zhao et al 2019). The insulin like growth factor (IGF-1) gene is the direct target of miR-323-3p (Wang et al 2019). The importance of different miRNA;s have started to be revealed and the significance of different miRNA:s in the treatment of ovarian dysfunction is just beginning to be realized.
THE PROPOSED STUDY
The positive animal experience of treatment of animals with induced PCOS is now progressing into the clinic. A review of the government website “clinicaltrials.gov” indicates that such studies have begun but no results have been presented.
Combining different sources of stem cells and stem cell products with PRP enhances the clinical response (Zhang et al 2019, Tandelwadka & Karthic 2020, Hsu et al 2021). In our study we plan to inject a combination of MSC, exosomes and PRP into the ovarian tissue in patients with PCOS to improve clinical symptoms and improve their chances to get pregnant. The safe use of the different components is well documented in the medical literature. The actual injection into the ovary brings with it the risk of bleeding, haematoma formations, infection, and inadvertent trauma to internal organs. However, intraovarian injection is similar to sonographic egg retrieval performed in IVF procedures which has been in use for 35 years and found to be extremely safe, but requires light anesthesia. At Friberg Fertility we have over 40 years of experience with IVF and can easily accommodate the intraovarian injection therapy.
The two human ovaries have a limited volume. We expect the be able to inject about 5 million stem cells, 0.5 ml exosomes, and 0.5 ml PRP into each ovary. A few minutes after the preparation of MSC, exosomes, and PRP it will be mixed with Selphyl® (see below) and injected into both ovaries. Selphyl® is a platlet-rich fibrin matrix created by conversion of fibrinogen to fibrin. When it is injected into the ovarian “niche” it works as a scaffold and keeps small molecules such as cell cytokins and growth factors in the intended location for a long time.
The intraovarian injection is performed in the early part of an induced or spontaneous menstrual cycle. Before the injection blood will be obtained for AMH, FSH, LH, E2, P4, Testosterone, Free Testosterone, DHEA-S, Androstenedione, and an anthral follicle count will be performed.
The patient is then followed with the same blood hormone levels every 2 – 4 weeks. It may take several months before PCOS symptoms and laboratory results become normal. Repeat ovarian infusion of the active ingredient is also a possibility in resistant patients. Then aggressive clomid, gonadotropin or IVF therapy is recommended. After ovarian injection of MSC in patients with ovarian failure or poor response in IVF several spontaneous pregnancies have occurred and therefore less invasive treatment or even simple observation can also be considered.
FDA and STEM CELL TREATMENT
The Federal Drug Administration (FDA) has for a long time maintained that stem cells are drugs and should be controlled by them. That means that pre-clinical and clinical testing should be performed and a proper license should be approved by the FDA. To obtain such a license for a new medication often expenses of $200 – $300 million dollars are required. Stem cell treatment is well established and it is highly unlikely that the pharmacological industry will be willing to obtain a FDA license for an approval of this type of treatment. Therefore treatments with stem cells have disappeared away from the US and has now mainly been located over seas and in the Caribbean. However, FDA:s opinion has been challenged in court and a recent legal verdict (September 2022) may bring back autologous (from your own body) stem cell treatment to the US.
Autologous stem cells are obtained from tissue extracted by liposuction from the abdomen, hips, or flanks of the patient. The tissue is processed in the “Time Machine”. Medikan, Kangnow, South Korea, which can produce a stem cell suspension the “stromal vascular fraction (SVF)” in about 2 hours. The SVF is very rich in stem cells. The process to obtain the SVF with adipose MSC is as follows.
Autologous Stem Cell Preparation From Fat Tissue:
- Patient will receive local anesthesia consisting of lidocaine 0.5% with epinephrine 1:400,00 with HCO3 8.4% titrated to pH of 7.4 (generally 5cc of HCO3 in total volume of 60cc)
- Patient undergoes sterile prep.
- Patient undergoes liposuction procedure utilizing the Time-Machine™ device, fat processing unit (syringe) and 2.5 – 3 mm cannula.
- Bacitracin ointment and a band aid are secured over the wound along with a compressive bandage.
- The ADSC-SVF with MSC are prepared in a closed system according to the following protocol:
- Lipokit harvest of fat into 60cc TP-101 syringe (single use sterile fat processing syringe)
- Centrifuge at 2800 rpm x 3 min.
- Remove free fatty acids and debris (local/blood) via TP-109 closed system
- Transfer 25cc of condensed fat to TP-102 syringe (SVF processing syringe)
- Add pre-warmed (38°) 25cc of Roche Liberase (collagenase formula will be labelled as T-Max Time Machine Accelerator) containing 12.5 Wunsch units.
- Incubate at 38°C for 30-45 minutes.
- Centrifuge at 200g X 4 minutes.
- Remove supernatant fluid leaving the bottom 3 – 10cc
- Add 50cc D5LR as a washing solution to remove collagenase residue and centrifuge at 200g X 4 minutes. Repeat 2 more times for a total of 3 washings.
- Remove all supernatant fluid leaving 3 – 10 cc of infranatant collection – this is the Stromal Vascular Fraction
- Transfer SVF to 10cc syringes through 100 micron filters.
- Cell sample collected and identified for number of cells, viability and to confirm no clumping or debris. Cell sample concentrated to approximately ml volume.
Allogenic (from another person) umbilical cord mesenchymal stem cells (UC-MSC) are obtained from Biopharma, Golden, Colorado. They are manufactured under strict environmental and laboratory control using cGMP (current Good Manufacturing Practice). After production UC-MSC are frozen in liquid nitrogen and sent to the Medical Surgical Association Clinic, St Johns in Antigua where the intraovarian stem cells infusion will be performed. Allogenic MSC use in the USA is still considered illegal and this type of treatment is still delegated to foreign counties and the Caribbean.
Exosomes are commercially available from Kimera or Vitti Laboratories. They are obtained from placental MSC of a very selected group of patients who are delivered by elective cesarean section. After review of patients medical history, extensive medical exams during the pregnancy and multiple testing using the FDA’s strict cell donor requirements protocol, their placentas are approved for further processing. Placental stem cells are extracted and cultured under low oxygen tension to produce an increased yield of exosomes. The actual placental MSC are then destroyed and removed. The fluid with the exosomes is concentrated using ultracentrifugation (>100,000 G) and tested for their content of a large number of growth factors and cell cytokins. They are thereafter frozen and kept at -20°C, which means they are easily transported (on e.q. frozen ice) and immediately ready for use after thawing.
The exosome possess a natural “homing in” ability and can travel long distances in the body. Due to their small size they are even capable to cross tight physiological barriers such as the blood/brain, blood/testis and blood/granulosa cell barriers. Exosomes are stable in the circulation and are undetectable by the immune system which make them viable for a longer time than MSC and helps with longer viability and existence in biological systems. It has even been found that exosome remain intact after being subjected to digestive enzymes because of their lipoprotein membrane envelope that sequesters them and their cargo from degeneration.
Exosomes have multiple direct positive effects in reproductive medicine and a few highlights can be mentioned. Exosomes are able to increase the blastomere count and release growth factors such as VEGF, PDGFAA in the developing blastocysts of murine embryos (Blazquez et al 2018). Furthermore, exosomes have been isolated from the uterine fluid and have been proposed to mediate endometrial-embryo cross talk at the time of implantation (Ng et al 2013).
A concern has occasionally been raised if growth factors and cell cytokins can stimulate cancer. These growth factors act on the cell membrane and not on the cell nucleus and can therefore not provoke cancer. Growth factors are normal cell constituents and activate internal cytoplasmic signal proteins and do not have mutagenic properties such as radiation, UV-light, tobacco, and certain chemicals.
Platlet-Rich Plasma (PRP) is prepared from the patients own blood. After centrifuging the platlet rich serum fraction, the platlets are activated to release growth factors (usually with calcium citrate). After activation PRP releases a large amount of cytokins and growth factors all aimed at tissue repair, growth and development. Among the growth factors in PRP it should be mentioned that growth differentiating Factor 9 (GDF9) is present in PRP. CDF9 is important for oocyte maturation and is important together with other growth factors to promote development of primary oocyte follicles into pre-anthral stages and this is reflected in increased AMH levels. The rejuvenating effect of PRP on the ovaries is now a well established (see reviews by Atkinson et al 2021, Rosario & Anderson 2021, Sharara et al 2021)
Keeping it all In Place
When small molecules like cytokins, cells, exosomes, and growth factors are prepared and injected they may easily disperse into the general circulation. In order to keep as large a portion as possible of the injected material in the ovaries, different scaffold substances have been used (Su et al 2016, Ding et al 2018). We have elected to use Selphyl® which creates a cross lattice as fibrinogen is converted to fibrin that effectively binds the molecules in place and in addition creates a fairly consistent PRP activation system. The usefulness of Selphyl® has been documented in multiple studies.
Preparation of PRP with Selphyl® PRFM
- A small amount of blood ( 9 ml) is obtained by venepuncture and placed in a vacuum collection system tube containing a cell separator gel.
- The tube is spun at 1,100 g for 6 minutes. The supernatant containing plasma and platelets is removed.
- Red and white blood cells are located under the cell separation gel.
- The plasma with platelets is placed in a second vacuum tube containing a small amount of calcium citrate.
- Polymerization to fibrin begins.
- The Sephyl® PRFM remain liquid for 20 minutes and should be injected during this time period.
- The platelet growth factors are released for up to 7 days or more.
There is a plethora of anecdotal and more recently evidence-based information to suggest MSCs and their exosomes may have significant beneficial use for a large variety of inflammatory, autoimmune, and degenerative conditions. They have been demonstrated to show immune-modulatory features mediated through T-regulatory cells. We believe it is important to demonstrate that there are minimal adverse events and that these are acceptable risks primarily related to the method of harvesting and deployment. Our closed system of production and final filtering of particles over 100 microns (using FDA approved nylon micro-filter), we are confidante that the protocol is safe as shown in multiple other studies. For the stem cell treatment, we utilize autologous mesenchymal stem cells. Autologous means that the cells are coming from the patient herself and not from anyone else. Cells or tissue from another person are called allogenic. An autologous source is always the safest, most efficient, least costly and does not result in any antibody response, immunological reactions, or any adverse anaphylactic changes.
The exosomes we use are obtained from Kimera Laboratories or Vitti Laboratories. They are extensively tested for sterility and the content of growth factors, anti-inflammatory and immunomodulating substances are known. They have been extensively used in medicine and over 100,000 patients have so far been treated with without adverse effects.
PRP has been used for over 20 years in clinical medicine to provide support for healing and repair of many body tissues. As a blood product derived from the treated patient it induces no untoward effects and can be safely injected. As with all injection procedures some patients may experience mild and temporary irritation, swelling, bruising, itching, discoloration, and tenderness at the injection site.
For years different research groups have used varying preparation and activation methods to obtain PRP. This has made it difficult to evaluate the effect of the different concentrations of growth factors and their clinical response. We have therefore chosen to use a well standardized preparation method for PRP using a fibrin matrix technique called Selphyl® PRFM, a platelet rich fibrin matrix scaffold that keeps the injected PRP within the boundaries of the ovary.
2 cc exosome (XoGLO®/ EV Pure +®) – Three times concentrated for the ovaries $2,000.00
Stem Cell Harvesting
- Stem cell harvesting procedure and preparation of stem cells $3,100.00
- Facility charge (1 Hour) $800.00
- Supplies $1,000.00
Blood draw and laboratory preparation of PRP for SEPHARYL® PFM solution $700.00
Deployment of Stem Cells, Exosomes, and PRP
- Transvaginal deployment of stem cells into ovaries using direct sonographic $2,000.00
- Anesthesia (30 Min) $400.00
- Facility charge. (30 Min) $500.00
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