Rejuvenation of Premature Ovarian Failure With Stem Cells

Premature ovarian insufficiency (POI), formerly referred to as premature ovarian failure
(POF), is defined as hypergonadotropic ovarian insufficiency occurring prior to age 40 (1).
It is surprisingly common and affects approximately 1% of women below the age of 40 (2, 3).
The incidence is 10% to 28% in women with primary amenorrhea and 4% to 18% in women with
secondary amenorrhea (2). The clinical manifestations include amenorrhea and abnormally high
levels of luteinizing (LH) and follicle stimulating hormone (FSH) and low levels of Anti
Mullerian Hormone (AMH). The etiology of POI is unknown in most cases. It can be caused by a
combination of inherited conditions such as immune disorders, environmental toxins and
iatrogenic injury (2, 3). The majority of patients with POI are considered to have idiopathic
premature ovarian insufficiency because usually no cause can be identified (3). POI has been
shown to be associated with an increased incidence of other conditions, including
Alzheimer's, cardiovascular and immune system diseases, metabolic syndrome, osteoporosis,
diabetes and cancer of reproductive organs. POI is characterized by loss of secondary
follicles, arrested folliculogenesis, decreased estrogen production, and infertility (3). The
mechanism of ovarian insufficiency is most likely accelerated follicular atresia but detailed
pathogenesis is yet to be fully understood (2).

In women younger than 40, at least two menopausal FSH levels (≥40 IU/L) will be sufficient
for the diagnosis of POI. It is not essential to have amenorrhea for the diagnosis of POI.
Subjects with oligomenorrhea or low ovarian reserve and elevated FSH, referred to as
transitional ovarian insufficiency (TOI), or at least 4 months amenorrhea together with FSH
levels exceeding 40 IU/liter, and are included in this category. Spontaneous menstrual cycles
can sometimes be seen after the diagnosis of POI as well. Resumption of normal ovarian
function, albeit temporary, in patients with normal karyotypes has been documented in 10 to
20% of patients; thus, spontaneous resumption of fertility is possible (1).

No therapeutic intervention has proven effective in restoring fertility in patients with POI.
Currently, egg donation remains the only reliable method to establish a pregnancy in women
with POI (2, 3). Even though this approach is attainable, resulting children will not be
genetically related to the recipient mother. Furthermore, egg donation is not ethically
acceptable to many couples. Various attempts at ovarian stimulation patients with POI are
usually unsuccessful because they are poor responders. Therefore, the diagnosis of POI can
cause great physical and mental suffering among these patients. Thus, there is critical need
to develop novel effective approaches for the treatment of POI.

Throughout life, there is an ongoing physiological level of atresia of oocytes. A decreased
germ cell endowment combined with an increased rate of germ cell destruction can explain POI
(1). The current concept that the ovary has a static ovarian reserve is entirely at odds with
the germ cell dynamics. Current research supports the concept that the ovary continues to
produce new germ cells into adult life, however, whether this occurs in humans is not
universally accepted. Recent research suggests that diminished ovarian reserve is a result of
the aging of the niche rather than a defect in the germ cells (4-6). Anti-mullerian hormone
has been used as a reliable biomarker for ovarian reserve in humans and the circulating AMH
levels change with age as follows:

20-25: 1.23-11.51 ng/mL 26-30: 1.03-11.10 ng/mL 31-35: 0.66-8.75 ng/mL 36-40: 0.42-8.34 ng/mL
41-46: 0.26-5.81 ng/mL 47-54: <0.82 ng/mL Emerging evidence suggests that bone marrow-derived
mesenchymal stem cells (BMSCs) could restore the structure and function of injured tissues
(7). During embryologic development, cells of the mesodermal layer give rise to multiple
mesenchymal tissue types including bone, cartilage, tendon, muscle, and fat and marrow stroma
(8). These precursor cells, also present in the postnatal organism, are referred to as
mesenchymal stem cells. These stem cells have been shown to retain their developmental
potential following extensive sub-cultivation in vitro. Implantation of culture-expanded
mesenchymal stem cells has been demonstrated to effect tissue regeneration in a variety of
animal models and depends on local factors to stimulate differentiation into the appropriate
phenotype (8).

Recent studies suggest that stem cell therapy holds promise in treatment of variety of
diseases including reproductive dysfunction. In a recent investigation by Ghadami et al
(2012) in our lab at Augusta University in Georgia, BMSC treated animals resumed ovarian
function (9). In another investigation by Lui et al (2014), granulosa cell apoptosis induced
by cisplatin was reduced when BMSCs were migrated to granulosa cells in vitro. In this study,
chemotherapy-induced POI rats were injected with BMSCs. The BMSCs treatment group's antral
follicle count and estradiol levels increased after 30 days, compared with untreated POI
group (10). In a recent clinical trial in Egypt, Edessy et al (2014) evaluated the
therapeutic potential of autologous mesenchymal bone marrow stem cells transplantation in
women with POI. Ten patients with POI were selected and their ovaries were injected with
autologous BMSC at time of laparoscopy. The results revealed resumption of menstruation in
one case after 3 months; two cases showed focal secretory changes after having atrophic
endometrium (11). According to these results, BMSC seem to have the ability to revive
prematurely failed ovaries both in their hormonal and follicular development abilities.

Furthermore, in this clinical trial, while at Augusta University, Georgia, USA, we have
successfully initiated two cases so far (8/10/2017). Both patients have tolerated the
procedure very well with no reported side effects or complications. Our first patient resumed
menses after 6 months post stem cell injection and she demonstrated decreasing levels of
serum FSH and increasing serum Estradiol levels (from undetectable levels pre-procedure, as
well as the one week, one month and 3 months' time points assessments to 96pg/ml at the 6
months' time point (latest data available so far). She also reported amelioration of her
post-menopausal symptoms including a decrease in hot flashes frequency and severity,
decreased vaginal dryness and improved sleeping patterns. The second patient will be finished
with the post-procedure follow up period on 2/21/2018.

Stem cell therapy has been shown to be beneficial and effective in various disease processes.
The safety of the stem cell therapy has been assessed in multiple clinical trials. Although,
autologous mesenchymal stem cell therapy for premature ovarian insufficiency is a novel
approach, the safety and success of such stem cell therapy has been demonstrated in many
other disease processes including graft versus host disease, acute myocardial infarction,
acute respiratory distress syndrome (ARDS) and many other diseases (12-14). There are
additional ongoing trials (Pilot -Phase III) to further assess risk and safety of stem cell
based therapies (15-17). This interventional pilot clinical study will investigate the use of
stem cell therapy to restore steroidogenesis, folliculogenesis, menstruation, and fertility
in participants.

Inclusion Criteria

- Signed and dated informed consent

- Female over the age of 18

- Diagnosis of premature ovarian insufficiency: At least two menopausal FSH levels (≥ 40
IU/L) and/or Primary or secondary amenorrhea at least for 3-6 months

- Diagnosis of low ovarian reserve defined as: AMH < _0.42 ng/ML & FSH >20 IU/L, and/or
failure of prior attempts of assisted reproductive techniques due to limited ovarian
response (poor responder).

- Normal karyotype 46, XX.

- Presence of at least one ovary

- Presence of at least unilateral tubal patency (with any clinically acceptable
methods),

- Acceptable uterine anatomy (by any clinically and/or imaging acceptable methods)

- No male infertility as assessed by normal semen analysis parameters and no sexual
dysfunction

- Normal thyroid function as evidence by normal serum Thyroid Stimulating Hormone (TSH)
levels, normal anti-microsomal antibodies.

- Agree to report any pregnancy to the research staff immediately.

- Willing and able to comply with study requirements and follow up instructions.

- Planning and/or willing to attempt to get pregnant as part of the experimental
protocol.

- No other causes of female infertility in the subject

Exclusion Criteria

- Currently pregnant or breast-feeding

- Has a history of, or evidence of current gynecologic malignancy within the past three
years

- Presence of adnexal masses indicating the need for further evaluation.

- Major mental health disorder that precludes participation in the study

- Active substance abuse or dependence

- Unfit or unwilling to undergo laparoscopy; has contraindication to laparoscopic
surgery and/or general anesthesia

- Current or recent (within the past 2 weeks) use of the following medications: Oral or
systemic corticosteroids, Hormones (estrogen, progestins, oral contraceptives),
Danazol, anticoagulants, herbal or botanical supplements with possible hormonal
effects. Washout will be allowed.

- Medical conditions that are contraindicated in pregnancy

- Type I or Type II diabetes mellitus, or if receiving antidiabetic medications

- Known significant anemia (Hemoglobin <8 g/dL).

- Untreated deep venous thrombosis, and/or pulmonary embolus

- Untreated cerebrovascular disease

- Known heart disease (New York Heart Association Class II or higher).

- Known Liver disease (defined as Aspartate Aminotransferase (AST) or Alanine
Aminotransferase (ALT)>2 times normal, or total bilirubin >2.5 mg/dL).

- Known Renal disease (defined as Blood urea nitrogen (BUN)>30 mg/dL or serum creatinine
> 1.6 mg/dL).