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RESEARCH, THEORY and  PRACTICE

Women eventually reach an age when their monthly cycles simply vanish. They are also far less likely to conceive as they get older.

This was well known back in Biblical days of parchment and papyrus, even if nobody really knew exactly why.

Now, DNA fingerprinting has shown embryo ploidy error in failed IVF cycles [1] to be the major dividend of older eggs over time.

This also helps explain chromosomal problems in offspring .. which go up as the mother’s age likewise increases.

While these challenges of “older ovaries” persist as our chief problem in IVF practice today, fresh plasma growth factors may at last bring a workable solution.

Could FertiGen be that answer? Can bad egg quality really be corrected? Despite switching IVF protocols–or even changing clinics–this usually achieves little when patient age goes above 40.

And why does advice on second IVF cycles seem so scattered? Will experts ever agree on a general strategy, a basic action plan to optimize egg recruitment?

Not really. This is because the remedy to the “old ovary” question remains largely unresolved. This lack of consensus actually enables an increasingly competitive, thriving pharma market supplying it all. 

Sadly, even when additional oocytes are retrieved in a later IVF cycle, egg quality usually remains poor.

In other words, simply getting more eggs from IVF doesn’t really matter if all the oocytes are of low quality anyway.

Yes it’s true that donor egg IVF offers a strong answer to this problem. By ‘borrowing’ oocytes from matched donors of low age who themselves have no infertility diagnosis, IVF success can be outstanding.

But imagine if there were a technique to dial back the odometer on the high-mileage ovary?  

Research has shown that older oocytes with chromosomal errors were more often harvested from follicles with the worst perfusion. This link between oxygen delivery and cell fitness was intriguing.

More research revealed that blood flow to the follicles can predict pregnancy rate in IVFOur group also began to see connections across chromosomal competency of embryos, molecular signaling, ovarian vascularity, and follicular oxygenation.

Because IVF drugs act downstream near the time of ovulation, FSH & LH shots alone seemed “too little, too late” to fix any preexisting low oxygen problems. 

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The tissue repair properties in general (and angiogenic effects in particular) associated with growth factors from platelet rich plasma (PRP) are well known, with wide use in other clinical contexts.

So, the basic concept which led to FertiGen was envisioned as something before IVF to improve reproductive outcomes. 

Why? Because any gains in ovarian blood flow should help oocyte competence by improving upstream cellular O2 and/or lowering concentrations of toxic intraovarian reactive oxygen species, well before any IVF medications can act on the follicle pool later.

Dr. Sills and colleagues have advanced two theories to explain observed outcomes after our fresh plasma techniques.

We published our oocyte perfusion model from Korea, while the second paper (Tehran) detailed the first known example of embryo ploidy rescue

This latter mechanism is more tenuous but may be a secondary effect of amplified oxygen delivery, by mRNA upregulation coordinated by molecular signaling from platelet-derived growth factors as noted in other tissue systems.

Links between ovarian oxygen status and egg competency gained better definition when perifollicular vascularity measured by color doppler ultrasound was correlated to intrafollicular dissolved oxygen content by analysis of follicular fluid obtained at egg collection for IVF. 

As you may know, during IVF egg retrieval fluid is obtained from each cyst (follicle) puncture along with the oocyte.

Typically this liquour folliculi is meticulously examined by microscope to identify, isolate, and catalogue each egg harvested, and any left-over follicle fluid is no longer needed & just discarded.

There is important information to be gleaned from this fluid, however. This is the material most close to the developing egg over time and can provide meaningful clues about the status of the oocyte.

Not surprisingly, ovarian perfusion closely matches intrafollicular O2 concentration – follicles with lower dissolved oxygen levels (in the fluid) more often produce eggs with cytoplasmic and chromosomal disorders [2,3].

Furthermore, this disruption appears to linger such that embryos most likely to implant originate from follicles that were well-vascularized and best oxygenated [2,3]. A hypoxic (“oxygen starved”) microenvironment thus puts an unwanted stress on ovarian function in general, and oocyte physiology in particular.

Allied research from gerontology, nutrition, lipid/free-radical chemistry, and regenerative medicine has given many insights into how this happens.

For example, low O2 partial pressure in follicular fluid of older and poorly perfused ovaries amplifies various local cell death pathways, ramping up apoptosis (cell death) and necrosis (decay).

When this happens, nutrient supply to the oocyte is curtailed and this deprivation results in the production of harmful reactive oxygen species (ROS), culminating in both granulosa cell and oocyte autophagy (self-digestion).

The corrosive impact of intrafollicular ROS is hard to overstate, extending to interference with meiotic spindle formation—which is notably attenuated and sometimes completely missing [9].

Experimental work with animal oocytes in vitro has shown oxidative stress is linked to altered DNA methylation, early cell death, and disordered meiosis and fertilization [10-12].

reprinted from WOOD & SILLS, 2020

ROS cut-point levels in follicular fluid have been calculated against key developmental benchmarks, and there seems to be a threshold value above which “viable embryo formation is not favorable” [13].

This means there is only so much stress the aging egg can tolerate before bad things happen. 

Even for patients not seeking pregnancy, that low-oxygen-ovary environment matters because it can still hasten onset of menopause [14].

Although the full reproductive pathology cascade from reduced vascular flow, limited ovarian perfusion, and insufficient follicular oxygen awaits additional study, any improvement in ovarian vascularity would be desirable.

It was against this background that FertiGen was developed.

While younger eggs do have a suite of molecular mechanisms to address oxidative damage and thus conserve germline fidelity, older oocytes show blunted clearance of reactive oxygen species, decreased DNA repair, reduced sensitivity of the spindle assembly checkpoint, and decreased capacity for protein repair and degradation [15].

So age-related ovarian hypoperfusion will just make matters worse, further hampering the older oocyte’s already enfeebled ability to mitigate such microstructural damage.

One relatively new and highly specialized clinical setting revealing the role of properly perfused ovarian tissue is among cancer patients who undergo surgical ovarian tissue reattachment.

Survival of ovarian tissue grafts appears to hinge on reestablishing adequate vascular support, and knowledge of how best to optimize the functional capacity of frozen-thawed ovarian tissue autografts is required for the operation to succeed.

Cryopreserved ovarian implants are expected to sustain considerable tissue ischemia until capillary perfusion is restored after thaw, an attrition causing significant follicular loss not unlike the benign ovarian aging noted in clinical IVF practice.

In 2013, successful adnexal injection of platelet-derived growth factors (using PRP) was reported for post-transplant neogenesis to boost viability of ovarian tissue autografts [16]. PRP was later shown to prevent ischemia and reperfusion damage [17].

Using platelet-derived growth factors to help improve circulation for surgically grafted ovarian tissue exactly parallels what is needed before IVF, where low oxygen perfusion is also the enemy.

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la science pour nouvelle vie

FertiGen

science for new life

Platelet products are closely involved in tissue regeneration, cell migration, extracellular matrix remodeling, programmed cell death, differentiation, and blood vessel proliferation [18].

At ovulation, platelets help coordinate local tissue repair from capsular microtrauma in the adult ovary and probably direct wider tissue remodeling effects, too [19].

Preliminary evidence from here and colleagues in Europe has shown intraovarian injection of autologous PRP can boost low ovarian reserve [20,21].

Our success with FertiGen could be explained by the intraovarian release of many soluble mediators triggering or facilitating angiogenesis including vascular endothelial growth factors (VEGF), basic fibroblast growth factor (FGF-2), platelet-derived growth factors (PDGFs), transforming growth factor (TGF), platelet-derived angiogenesis factor (PDAF), epidermal growth factor (EGF), as well as several interleukins & insulin-like growth factors [22,23].

But if the ovarian perfusion dynamic attained by this array of platelet-derived growth factors merely amplifies the number of crappy eggs retrieved from aged ovaries, then nothing relevant is really gained when genetic imbalances persist in all oocytes collected.

There was nothing to gainsay this critique until our research suggested platelet-derived growth factors could enable “ploidy rescue” for human embryos, and a healthy baby boy was delivered at term following that intervention [24].

It is now hypothesized that injection of autologous growth factors with FertiGen places angiogenic inputs to dormant ovarian tissue, and the associated uptick in perfusion increases not only the available oxygen, but also delivers an array of genetic regulatory signals, either to ovarian stem cells or to latent, senescent oocytes.

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Either pathway would be welcome. Each mechanism could be useful to correct upstream anomalies in the oocyte-to-embryo transition, including ovarian stem cell differentiation, mitochondrial dynamics, mRNA storage, translation, and degradation.

Further study is underway to show how cytokines produced by activated platelets impact chromosomal competency during oogenesis. Related research in dental surgery has established PRP-promoted cell-specific migration, proliferation, and  differentiation after upregulation of Scx in oral target tissue [25].

Gene expression patterns specific for tissue maturation were mimicked in PRP treated cartilage, with downregulation of Collagen Types II and X, while deiodinase II and netrin-1 were upregulated [26]. In addition, PRP has been implicated as a regulatory mediator for CCAAT/enhancer binding protein β and E2F transcription factor 1 (E2F1) [27]. Corollary work with adult human ovarian tissue will help clarify the actions of platelet derived growth factors on oocyte genetics.

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reversing aging effects

FertiGen

umkehrung der auswirkungen des alters

Older ooplasm generally contains fewer mitochondria, and older eggs have impaired fertilization and the embryos to which they contribute show poor development—perhaps in part due to an altered mitochondrial function [28].

Of note, a relatively high mtDNA copy number is often seen among aneuploid embryos [29] and those with elevated mtDNA copy number have a lower chance of producing an ongoing pregnancy [30].

If one gain from enhanced oxygen perfusion after FertiGen is better ooplasm quality, then subsequent recovery of mitochondrial function could be another pathway for ploidy rescue of blastocysts.

These are major issues to restore fertility, yes. But for every one woman who needs reproductive help there may be 8 or 10 who seek regenerative support more generally. As noted in Ovarian Reboot, when the ovary gets proper oxygen, the benefits appear to go much further:

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final thoughts

FertiGen

schlussfolgerung

The full range of factors causing ovarian insufficiency may never be known at the molecular level, but oxygen perfusion likely plays an outsized role. If our perfusion theory is valid, then this would go a long way to explain why some FertiGen patients respond so favorably to the procedure.

Consider women who smoke and how they experience menopause earlier than matched non-smoking controls. This confirms the catastrophic results of oxygen starvation for the ovary [31].

Our approach with FertiGen aims to meet this problem with a view to fix a crucial deficiency, ultimately to diminish the need for donor oocytes as well as conventional hormone replacement therapy [32].

Follicular vascularity and oxygen content in the microenvironment closest to the developing oocyte are increasingly recognized as critical determinants of egg competence. Insufficient oxygenation from dampened follicular perfusion results in oxidative stress and tissue injury. As discussed earlier, this translates into widespread developmental problems throughout the oocyte.

The angiogenic (capillary stimulating) features of the platelet derived cytokines with FertiGen may assuage ovarian hypoxia by facilitating improved vascularity concurrent with correction of dysregulated gene function.

If the stage is set properly with documentation of improved ovarian reserve after treatment, we believe that FertiGen can bring reproductive outcomes within reach that were once thought impossible – including healthy term livebirths.

Down-regulation of genes controlling DNA repair and damage response pathways may result from upstream hypoperfusion injury even before birth. The developing ovary is highly sensitive to such gestational hypoxia, having implications for future fertility in next-generation offspring [33-35]. Thus, exposure to very low O2 conditions during fetal development may hasten ovarian aging and decrease reserve later in adulthood.

While synthetic gonadotropins used for follicular recruitment are an important component of the advanced reproductive technologies, the shots women must take during IVF are better thought of as pharmacologic brushstrokes on a blank ovarian canvas. Platelet derived growth factors with FertiGen work to prepare the ovaries to optimize controlled ovulation induction later.

Additional investigation focusing on the ovary (including ovarian stem cells) is needed to define which platelet-derived signals are most involved in oocyte replenishment and “ploidy rescue”, thereby offering new methods to make IVF successful for those patients with the lowest ovarian reserve. Further studies of the ovarian germline stem cell niche and its requisite regulatory inputs promise to yield valuable insights into reproductive aging.

Your interest in our clinical program and FertiGen® is appreciated; I hope the information shared here has been useful. It would be an honor to answer any additional questions and have an opportunity to help.

   – Dr. Scott Sills

References

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2. Van Blerkom J. Epigenetic influences on oocyte developmental competence: perifollicular vascularity and intrafollicular oxygen. J Assist Reprod Genet 1998;15(5):226-34.

3. Borini A, Maccolini A, Tallarini A, Bonu MA, Sciajno R, Flamigni C. Perifollicular vascularity and its relationship with oocyte maturity and IVF outcome. Ann NY Acad Sci 2001;943:64-7.

4. Fernandez-Moure JS, Van Eps JL, Cabrera FJ et al. Platelet-rich plasma: a biomimetic approach to enhancement of surgical wound healing. J Surg Res 2017;207:33-44.

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9. Chattopadhayay R, Ganesh A, Samanta J, Jana SK, Chakravarty BN, Chaudhury K. Effect of follicular fluid oxidative stress on meiotic spindle formation in infertile women with polycystic ovarian syndrome. Gynecol Obstet Invest 2010;69(3):197-202.

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13. Jana SK, K NB, Chattopadhyay R, Chakravarty B, Chaudhury K. Upper control limit of reactive oxygen species in follicular fluid beyond which viable embryo formation is not favorable. Reprod Toxicol 2010;29(4):447-51.

14. Yadav AK, Yadav PK, Chaudhary GR et al. Autophagy in hypoxic ovary. Cell Mol Life Sci 2019 May 6. doi: 10.1007/s00018-019-03122-4.

15. Mihalas BP, Redgrove KA, McLaughlin EA, Nixon B. Molecular Mechanisms Responsible for Increased Vulnerability of the Ageing Oocyte to Oxidative Damage. Oxid Med Cell Longev 2017;2017:4015874.

16. Callejo J, Salvador C, González-Nuñez S et al. Live birth in a woman without ovaries after autograft of frozen-thawed ovarian tissue combined with growth factors. J Ovarian Res 2013;6(1):33.

17. Bakacak M, Bostanci MS, İnanc F et al. Protective Effect of Platelet Rich Plasma on Experimental Ischemia/Reperfusion Injury in Rat Ovary. Gynecol Obstet Invest 2016;81(3):225-31.

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21. Sills ES, Rickers NS, Li X et al. First data on in vitro fertilization and blastocyst formation after intraovarian injection of calcium gluconate-activated platelet rich plasma. Gynecol Endocrinol 2018;34(9):756-60.

22. Lubkowska A, Dolegowska B, Banfi G. Growth factor content in PRP and their applicability in medicine. J Biol Regul Homeost Agents 2012;26(2 Suppl 1):3S-22S.

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24. Sills ES, Rickers NS, Svid C, Rickers JM, Wood SH. Normalized ploidy following 20 consecutive blastocysts with chromosomal error: Healthy 46,XY pregnancy with IVF after intraovarian injection of autologous enriched platelet-derived growth factors. Int J Mol Cell Med 2019;8(1):84-9.

25. Imai S, Kumagai K, Yamaguchi Y, Miyatake K, Saito T. Platelet-rich plasma promotes migration, proliferation, and gene expression of scleraxis and vascular endothelial growth factor in paratenon-derived cells in vitro. Sports Health 2019;11(2):142-148.

26. Zhang Y, Morgan BJ, Smith R et al. Platelet-rich plasma induces post-natal maturation of immature articular cartilage and correlates with LOXL1 activation. Sci Rep 2017;7(1):3699.

27. Shen H, Cheng H, Chen H, Zhang J. Identification of key genes induced by platelet-rich plasma in human dermal papilla cells using bioinformatics methods. Mol Med Rep 2017;15(1):81-88.

28. Sills ES, Takeuchi T, Tucker MJ, Palermo GD. Genetic and epigenetic modifications associated with human ooplasm donation and mitochondrial heteroplasmy—considerations for interpreting studies of heritability and reproductive outcome. Med Hypotheses 2004;62(4):612-7.

29. Jing Y, Li L, Li YY et al. Embryo quality, and not chromosomal nondiploidy affects mitochondrial DNA content in mouse blastocysts. J Cell Physiol 2019;234(7):10481-8.

30. Lledo B, Ortiz JA, Morales R et al. Comprehensive mitochondrial DNA analysis and IVF outcome. Hum Reprod Open 2018;2018(4):hoy023.

31. Whitcomb BW, Purdue-Smithe AC, Szegda KL et al. Cigarette smoking and risk of early natural menopause. Am J Epidemiol 2018;187(4):696-704.

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33. Oliveira PH, Boura JS, Abecasis MM, Gimble JM, da Silva CL, Cabral JM. Impact of hypoxia and long-term cultivation on the genomic stability and mitochondrial performance of ex vivo expanded human stem/stromal cells. Stem Cell Res 2012;9(3):225-36.

34. Aiken CE, Tarry-Adkins JL, Spiroski AM et al. Chronic gestational hypoxia accelerates ovarian aging and lowers ovarian reserve in next-generation adult rats. FASEB J 2019;33(6):7758-66.

35. Pampanini V, Jahnukainen K, Sahlin L et al. Impact of uteroplacental insufficiency on ovarian follicular pool in the rat. Reprod Biol Endocrinol 2019;17(1):10.

Reflect. Renew. Rejoice.