Jan Friberg, MD PhD
The IVF procedure used today was developed in the 1970s and 80s with a very low success rate but in the 1990s a respectable 25% pregnancy rate per started procedure indicated how quickly major progress was achieved. Today the success rate has improved to about 40% and a live birth rate of 80-90% can be expected after 3 egg retrievals in regular IVF patients, but this may not be a realistic figure for patient’s with fibroids, endometriosis, adenomyosis, or severe male factor infertility as well as older patients.
Patients labelled as “Poor Responders in IVF” present a very real challenge for the clinician. This particular group is defined as a patient with low Anti Mullerian Hormone (AMH) level of less than 0.5 – 1.1ng/ml, 5 or less preantral follicles Antral Follicular Count (AFC) seen on sonogram in the early part of the menstrual cycle and fewer than 3 eggs retrieved per stimulation cycle. This group consists of about 10% of all IVF patients aged 35-40 years and older, but this situation may also be seen in quite young patients.
Patients with a poor IVF response are often offered various “add ons” which may be used together with the ovarian stimulation medication such as clomid, letrozole, or growth hormone (Hart 2022) or as pre-treatment before IVF with Testosterone, Dehydroepiandrosterone-sulfate (DHEA-S), coenzyme Q10, HCG, LH, Estradiol, gonadotropin, releasing hormone agonists (GnRHa)and antagonists as well as oral contraceptive pills but no impressive improvements in pregnancy rates have been recorded (Kyrov et al, 2009).
STEM CELLS
Now stem cells, or stem cell products appear to give new hope for poor responders. Recruitment and development of ovarian follicles is a long process and has been estimated to take between 3 and 12 months depending on the location of the follicle within the ovary. Granulosa cells and theca cells in the ovary co-operate with the production of growth factors and cytokines to advance the oocyte to be ready for ovulation. FSH and LH are needed for completion of growth and release of the oocyte and other substances such as prostaglandins, steroids, and proteins are also involved. Growth factors also influence the meiotic spindle to ensure normal development and separation of the chromosomes at cell division, a function that is compromised in older oocytes. Growth factors and cytokines need to be present throughout the entire oocyte development and the gonadotropin stimulation in the IVF cycle is present for only a short time before egg harvest and this time is to short to have any profound effect on egg quality. Applying growth factors long before fertility stimulation starts seems to improve egg competence and indirect evidence also indicates that chromosomal abnormalities can be corrected. (Sills et al 2019).
Over the past 20 years stem cells and stem cell derived growth factors have been found to play an important role in various aspects of regenerative medicine. Different sources of stem cells have been investigated but mesenchymal stem cells (MSCs) have given the most promising results. MSCs from bone marrow, fat tissue, amniotic fluid, amnion, menstrual blood, umbilical cord blood and placental tissue all have the potential to renew themselves, have low immunogenicity and can differentiate into cells of the reproductive system. These MSCs secrete a large number of growth factors and cell cytokines directed to cell regeneration and differentiation. Paracrine signaling from these MSCs represent an important function that promotes anti-inflammation, immunoregulation, anti-apoptosis (prevent cell death), and antifibrosis as well as controlling local oxidative stress. Another important ability of MSCs is the capacity of “homing in”. Chemoattractants are released from damaged cells anywhere in the body and promotes accumulation of MSCs, exosomes, and PRP, to the damaged site and help to promote repair and recovery (Liu et al 2014). A simple way to obtain MSC is to extract them from adipose tissue (fat) obtained during liposuction.
Exosomes
Exosomes are small vesicles secreted from all body cells but exosomes secreted from stem cells have special significance. They contain the same growth and differentiation factors that are present in MSCs and can be obtained with a special technique from the growth medium in which the stem cells are cultured. Exosomes have benefits over MSC with characteristics such as smaller size, lower complexity, lack of nucleus (thus have no capacity for neoplastic transformation), increased stability, easier to handle, have longer presence in the circulation, and potential for loading and delivery of bio molecules. After collection they can be frozen on dry ice, shipped and are ready for use immediately after thawing. Micro RNAs (MiRNA) secreted in the exosomes have special functions on cell differentiation (Gangaraju & Lin, 2009)
PRP
Platelet rich plasma (PRP) is derived directly from the patients own blood. The circulating platelets contain a concentrated source of different growth factors and cytokines such as vascular endothelial growth factor (VEGF), Insulin like growth factor I (IGF-1), and 2 (IGF-2), epidermal growth factor (EGF) transforming growth factor (TGF-beta), and many others. Growth factors in platelets are inactive in the blood circulation, but are activated after tissue trauma. For clinical use the platelets can also be activated by several laboratory procedures and used for regenerative treatment.
Animal studies
Early use of stem cells in reproductive medicine got started little over 15 years ago. These studies were focused both on the ability of MSCs to prophylactically protect germ cells that are exposed to gonadotoxic substances and the ability of stem cells to regenerate the germinal epithelium after the use of toxic treatment by inhibiting apoptosis and enhance gamete function (Liu et al 2007, Fu et al 2008, Abd-Allah et al 2013, Kilic et al 2014, Fouad et al 2016, Song et al 2016). Differentiation of MSC into germ cells or germ cell like cells have repeatedly bene documented (Hua et al 2009, Amidi et al 2015, Yan et al 2015, Hou et al 2016. Shlush et al2017, Ling et al 2019). Retinoid Acid as well as growth factors BMP4, BMP8b, FGF2, LIF and TGF beta support the differentiation of stem cells into germ cells and reduce apoptosis through up regulation of the anti-apoptotic gene B-cell lymphoma-2 (Fu et al2008, Abd-Allah et al 2013). Differentiation of MSCs into germ cells has also been promoted with the use of co-culture conditions (Cui et al 2009, Liu et al 2014?, Asgari et al 2015). Fertility of the created gametes have also been shown (Badawy et al 2017, Mohammed et al 2019)
With these and other extensive studies (reviewed by Esfandyari et al 2020, Sharara et al 2020, Chang et al 2021, Rosario & Anderson, 2021), it is clear that MSCs have the potential to become a new instrument for correction of ovarian malfunction.
Human Studies
In spite of the large amount of studies of MSCs performed on animals in regenerative reproductive medicine relatively few studies have been performed in humans. Edessy and co-workers (Edessy et al 2016) first reported the use of MSCs (from bone marrow) to treat patients with premature ovarian failure (POF), a condition previously known as premature menopause, the most advanced sign of ovarian dysfunction. Out of 10 patients injected with MSCs into their ovaries, two resumed menstrual cycles and one patient conceived naturally and had an uneventful delivery. More case reports and small series of patients with POF treated with MSCs have now been reported (Gabr et al 2016, Ding et al 2018) Yan et al (2020), Mashayekhi et al (2021). In many instances simple follow up was undertaken but in some instances IVF was used and pregnancy rates of 0-14% were reported (Mawet et al 2021). Perhaps a more aggressive gonadotropin stimulation could have given a higher pregnancy rate as reported by Ferreri et al (2020) who started multiple sequential gonadotropin stimulation cycles directly after surgical ovarian fragmentation with autologous ovarian tissue transplantation in POF patients and reported 4 pregnancies in 7 oocyte retrievals from a group of 14 POF patients recruited for the study.
MSCs have also been used to improve the success rate in patients with “poor response in IVF”, but the studies are few. Herraiz et al (2018) infused bone marrow MSCs into the left ovarian artery and noted an improved ovarian function of a small group of 16 patients and produced 5 pregnancies, 3 were spontaneous and 2 were obtained with IVF. After the MSC infusion into the ovaries, some patients had dramatic increases in AMH levels recorded but in the overall review of their patients, no significant increases in the AMH levels could be noticed. Tandulwadkar and Karthick (2020) combined bone marrow MSC and PRP and achieved significant improvement in antral follicle count and mature oocytes as well as good quality embryos. AMH increased in some patients but did not reach statistical significance.
It is surprising that not more studies have been performed with the use of the placental derived MSC since these exosomes come from young stem cells that produce at least 30% more exosomes than stem cells derived from older or diseased tissue. (Sun et al 2007, Carrion et al 2010, Chen et al 2011, Collins et al 2014). It has also been shown that injected MSCs accumulate in the liver and lungs and have a short lifespan. The exosomes from the MSCs survives much longer and is the most important mediator of clinical improvement. (Phinney & Pittenger 2017)
PRP has become increasingly popular for ovarian rejuvenation but is also used to improve the thickness of an abnormally thin endometrium. In reproductive medicine PRP was first used for its VEGF content to support ovarian tissue transplantation to a woman without ovaries. She had a successful procedure that resulted in the birth of a healthy baby (Callejo et al (2013). Pantos et al (2016) used intraovarian PRP injections to re-establish menstrual cycles in post-menopausal patients and also were able to document spontaneous pregnancies with this method. Several similar reports followed (Sfakianoudis et al 2019, Farimani et al 2019, Stojkovska et al 2019, Cakirogu et al 2020, Hsu et al 2020, Petryle & Petryl 2020, Hsu et al 2021) nicely summarized by Sharara et al (2021). An interesting report was presented by Melo and his group (Melo et al, 2020) where they injected PRP into the ovaries of patients with elevated baseline FSH levels. The 46 women treated with PRP showed significant improvement in FSH, AMH, and AFC (Antral Follicle Count) compared to 37 women in the control group who did not undergo any intervention. They recorded 13 pregnancies in the treatment group and 2 were obtained in the control group. In preparation for the 2021 ESHRE (European Society for Human Reproduction and Embryology) meeting Pellicer (2021) reviewed the world literature and reported a 28% live birth rate after ovarian PRP injection in patients with a poor response in IVF and a 10% pregnancy rate among patients with POF. He also suggested that a combination of stem cells and PRP appears to give better results than PRP alone.
Eligibility
Patients for this study will be recruited from the private practice of Friberg Fertility. They should have undergone several treatments with Human gonadotropins in order to get pregnant, preferably through an IVF procedure. They should also be documented to be “Poor ovarian Responders, (POR)” using criteria established by the European Society of Human Reproduction in Bologna 2010 (Ferraretti et al 2011). These criteria are as follows and at least two of the three features must be present:
- Advanced Maternal Age (>- 40 years) or any other risk factor for POF.
- A previous POR (less than 3 oocytes with conventional stimulation protocol
- An abnormal ovarian reserve test (I,e, AFC <5-7 follicles of AMH<0.5-1.1
A novel predictive model has been designed by the POSEIDON Group (Esteves et al 2018)to estimate the number of mature oocytes required to obtain at least one euploid embryo in different patient groups.. Using the POSEIDON criteria patients are divided into 4 groups:
| Group I < 35 years old AFC≥5 AMH > 1.2 ng/ml | Group II >35 years old AFC≥5 AMH ≥1.2 ng/ml |
| Group III < 35 years old AFC < 5 AMH < 1.2ng/ml | Group IV ≥ 35 years old AFC < 5 AMH < 1.2 ng/ml |
Group III and IV are eligible for this study.
The Planned Study
The purpose of this study is to evaluate if stem cells, placental derived exosomes, and PRP can improve the reproductive performance in the patient with “poor response” in IVF or “POF”. The end points will hopefully show an increase in quality and quantity of oocytes and embryos with increased pregnancy rates after infusion of the substances into the ovaries. Results seen before the ovarian injection will be compared to findings obtained after the ovarian injection.
Treatment
The first visit will introduce the couple to the background knowledge of both animal and human experience of ovarian rejuvenation and the use of stem cells and stem cell products. In the early part of a subsequent menstrual cycle the wife will have blood drawn for AMH, FSH, LH, Estradiol, Progesterone, and the ovaries will be examined for their antral follicular count (AFC).
The initial intervention is a liposuction under local anesthesia to obtain 50 grams of adipose (fat) tissue. This tissue is processed in the ”Time Machine” (Medican, Kangow, South Korea) to obtain the Stromal Vascular Fraction which is rich in MSC. This takes about 2 to 2 ½ hours. The stem cell fraction, exosomes, and PRP is then mixed using Selphyl® PRFM as described under “keep it in place”. The injection must be performed within 10 minutes of the mixing of the ingredients otherwise the solution solidifies. The technique to obtain the Stromal Vascular fraction is as follows:
- 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.
Conscious sedation is then started by an anesthesiologist. With the use of transvaginal sonography the ovaries are visualized and a special needle with a sonolucent tip is inserted under direct visualization into the left and right ovary. A 0.5 mL solution containing 5 billion autologous adipose tissue-derived stem cells form the Stromal Vascular Fraction or 0.5 mL allogenic stem cells, 0.5mL exosomes and 0.5ml PRP prepared with the Selphyl® technique. If an additional procedure needs to be performed, such as an evaluation of the uterine cavity or removal of polyps in the uterus, it can be performed at the same time. The patient rests in the post-op area for 30-60 minutes after the procedure, which is expected to last less than 10 minutes.
In the next menstrual cycle, and IVF stimulation is recommended using the same stimulation protocols that were used in the previous cycle when it was determined that the patient belonged to the “Poor responders group”. Pregnancies have occurred in unstimulated cycles after the ovarian rejuvenation, although experiences are limited. This observational approach appears to give a lower pregnancy rate than if the cycle is IVF Stimulated.
In the next menstrual cycle, an IVF stimulation is recommended using the same stimulation protocols that were used in the previous cycles when it was determine that the patient belonged to the “poor responder” group. Pregnancies have occurred in unstimulated cycles after ovarian rejuvenation so simple observation is an alternative.
OVERVIEW OF TREATMENT
Cycle I
Menstrual Day
2-5 – Blood for FSH, LH, Estradiol, Progesterone and AMH. A sonogram is also performed to evaluate the Antral Follicle Count (AFC). This is followed by Liposuction to collect the autologous Adipose Derived Stem Cell-Stromal Vascular Fractions (ADSC-SVF) under local anesthesia. The Adipose tissue will be processed in the “Time Machine” as described under “Autologous MSC” to obtain the stem cell rich SVF Fraction.
- MSC, exosomes, and PRP prepared with the Selphyl® technique.
- Conscious sedation is started and both ovaries are infused with the ADSC-SVF fraction. The procedure will take less than 10 minutes.
- Post-op the patient remains for 30 to 60 minutes for observation in the surgical unit before she is sent home.
Cycle II
2-3 – Blood for FSH, LH, Estradiol, Progesterone, and AMH. Baseline Sonogram performed; IVF is recommended for activation of secondary follicles. If a good response is obtained, perform the IVF. If the response is poor, take a rest cycle.
Cycle III
2-30 – If poor response, rest cycle
Cycle IV to VI and on ?
2-3 Repeat IVF Stimulation to activate primary and secondary follicles.
Keep it in place
Small molecules such as growth factors, cell cytokines, exosomes, and PRP components tend to disappear into the general circulation when deposited in the ovarian “niche”. To prevent this from happening scaffold material such as collagen fiber type 1 has been used to confine the injected particles to the ovarian “niche”. The effectiveness of this appeared been demonstrated in both animal studies (Su et al 2016) and in women (Ding et al 2018). We have elected to use Selphyl® PRFM because of its extensive use in clinical medicine. Selphyl® PRFM activate fibrinogen to create a fibrin clot. This coagulate binds the injected factors in place for a long time. Selphyl® PRFM is a natural product from the patient’s own blood, has no risk for allergic reactions , has a physiological pH , and has not reported side effects, except for the trauma associated with the injection procedure (Goldfarb & Shapiro 2012, Roy et al 2011, Sclafani 2011).
Preparation of PRP with Selphyl® PRFM
- A small amount of blood ( 9 ml) is obtained by venipuncture 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.
Safety
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.
Economical Considerations
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
$4,900.00
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
Observation.
- Anesthesia (30 Min) $400.00
- Facility charge. (30 Min) $500.00
Total $3,600.00
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