Stem cells restored ovarian function and folliculogenesis following cyclophosphamide-induced ovarian failure in rats.INTRODUCTION
Premature ovarian failure (POF) is a heterogeneous syndrome characterized by lack of folliculogenesis and ovarian estrogen production, associated with secondary amenorrhea and infertility in women under the age of 40 years. (1) The syndrome is represented in 1% of menopausal women (2-3), and in 0.1% under the age of 30 years. (4) POF may be idiopathic in 74-90% (5) or associated with genetic (6), metabolic (7) or autoimmune diseases. (8) However, iatrogenic causes following surgery, radiotherapy or chemotherapy are also recognized causes for POF. (4) Although fertility restoration is a significant concern for these women,ovarian failure is also associated with an increased risk of osteoporosis, cardiovascular disease and dementia. (9) Hormone deficiency can be overcome by hormone replacement with all its due concerns. (10) Nevertheless, the loss of fertility is a major health problem that affects these patients; currently no treatment is available that effectively restores fertility potential. (11)
Although return of spontaneous ovulation and conception has been recognized in women with POF (12-13), the use of donor eggs with assisted conception and/ or adoption is the only means of parenthood if spontaneous return of ovulation does not occur. These options are not accepted universally on moral, religious and social grounds. On the other hand, cryopreservation of ovarian tissues and/ or oocytes of patients before chemotherapy or irradiation for future fertilization should be offered before the start of treatment if future fertility is to be preserved. (14) Although early menopause frequently occurs in women with leukemia after chemotherapy, bone marrow transplantation has been linked to an unexplained return of ovarian function and fertility in some survivors. (15)
The aim of this study was to explore the therapeutic potency of MSCs (mesenchymal stem cells) transplantation on ovarian function and folliculogenesis for chemotherapy-induced ovarian failure in rats.
MATERIALS AND METHODS
This study was a prospective case control animal study in collaboration between National Research Centre of Cairo, Egypt (the Reproductive Health Research, the Medical Biochemistry Department, the Histology, Histo- Chemistry, and Pathology Department and the Animal Reproduction and Artificial Insemination Department), and the Faculty of Medicine of Cairo University (the Obstetrics and Gynecology Department and the Unit of Biochemistry and Molecular Biology of the Medical Biochemistry and Molecular Biology Departments). The bioethical committee of the National Research Centre approved the study (No. 08123).
Sixty mature white albino female rats were included in the study; their age at the beginning of the experiment was 60-70 days and their weight range was 200-250 mg. The rats were fed by rat chow and water (ad libitum) under controlled temperature (28-30[degrees]C) and relative humidity (50-60%). They were observed for 14 days prior to commencing treatment to ensure adequate adaptation.
Induction of Ovarian Failure
Fifteen rats were chosen randomly as a control group (Group I), while the other 45 rats (the Study Group) were administered with 50 mg/kg of CTX with intra-peritoneal (IP) injection, as a loading dose followed by daily IP dose of 8 mg/kg CTX for 14 consecutive days. (16) Vaginal smear was carried out daily at 10:00-11:00AM to ascertain ovarian failure. The cycles were regarded as normal when they showed typical stages of proestrus, estrus, met-estrus and di-estrus stages, which normally lasts for 4-6 days. The appearance of cornified cells was used as an indicator of estrogenic activity. (16) Biweekly hormonal profile of serum E2 and FSH estimation by Enzyme Linked Immuno- sorbent Assay (BioSource [TM] ELISA kits, USA) were carried out until ovarian failure was confirmed. This was achieved after two weeks of daily CTX.
Two rats were sacrificed at the end of the protocol for histopathological examination to confirm ovarian failure.
Preparation of Bone Marrow-derived MSC
Bone marrow was harvested by flushing the tibiae and femurs of 6-week-old male white albino rats with Gibco- BRL DMEM medium (Dulbecco's Modified Eagle's Medium, from Gibco[R]) and was supplemented with 10% fetal bovine serum (GIBCO/BRL).
Nucleated cells were isolated with a density gradient over Ficoll/Paque ( Pharmacia Fine Chemicals) and re-suspended in complete culture medium supplemented with 1% penicillin-streptomycin (Gibco-BRL). Cells were incubated at 37[degrees]C in 5% humidified CO2 for 12-14 days as primary culture or upon formation of large colonies.
When large colonies developed (80-90% confluence), the cultures were washed twice with PBS (Phosphate buffered saline) and the cells were trypsinized with 0.25% trypsin in 1mm EDTA (Gibco-BRL) for 5 min at 37[degrees]C. After centrifugation, cells were resuspended with serum-supplemented medium and incubated in 50 [cm.sup.2] culture flask (Falcon).
The resulting cultures were referred to as first-passage cultures. MSCs in culture were characterized by their adhesiveness and fusiform shape, detection of CD29 surface marker by PCR, and by their ability to differentiate into osteocytes and chondrocytes (data not shown).
Injection of Stem Cells
After confirming ovarian failure, the study group was divided into 3 equal groups, randomly (II, III and IV). Each group consisted of 15 rats. Rats of group II were injected intravenously once by male MSCs ([5x10.sup.6] cells) in 20-[micro]l saline, while those of group III were injected by 20-ul PBS only. The rats of group IV did not receive any injection.
This was carried out by daily vaginal smear at 10:00-11:00AM after completion of CTX injections and for eight weeks following the intervention to check for the number of normal estrous cycle changes.
In addition, fortnightly serum E2 and FSH estimation was carried out to assess the changes in ovarian function. Two rats were sacrificed from each group randomly at the end of the study to observe the resurrection of ovarian activity (follicular growth and count).
PCR detection of male-derived MSCs
Genomic DNA was prepared from ovarian tissue homogenates of the rats in each group using Wizard[R] Genomic DNA purification kit (Promega, Madison, WI, USA). The presence of Sry gene expression in the recipient female rats was assessed by PCR. Primer sequences for Sry gene (forward 5'-CATCGAAGGGTTAAAGTGCCA-3', reverse 5'-ATAGTGTGTAG-GTTGTTGTCC-3') were obtained from published sequences (17) and amplified product of 104bp. The PCR conditions were as follows: incubation at 94[degrees]C for 4 min; 35 cycles of incubation at 94[degrees]C for 50s, 60[degrees]C for 30s, and 72[degrees]C for 1 min; with a final incubation at 72[degrees]C for 10 min. PCR products were separated using 2% agarose gel electrophoresis and stained with ethidium bromide.
Data were statistically described in terms of range, mean value[+ or -] standard deviation. Comparison of quantitative variables was carried out using Wilcoxon signed--rank test for paired (matched) samples; whilst categorical variables were compared using Fisher's exact test with significance established at a two -tailed P value of <0.05.
The mean FSH [+ or -]SEM and E2 [+ or -] SEM values of the groups involved in the study are shown in Tables 1 and 2.
In the control group (Group I), the mean FSH and E2 levels were nearly constant all over the entire period of study. While in the study groups II, III and IV there were noticeable increase in the mean FSH level and decrease of mean E2 from the second week after induction of failure. This hypergonadotropinaemia and hypoestrogenic state was constant on follow up in Groups III and IV in spite of injection of normal saline for the Group III. Though, Group II showed an evidence of failure by mean FSH level of 8.39 [+ or -] 0.26 mIU/ml and mean E2 level by 32.387[+ or -]0.51pg/ml on the day of injection of stem cells, there was an abolishment of FSH rise and E2 decline thereafter. By the 8th week there was a return of mean FSH and mean E2 levels (5.38 [+ or -] 0.31mIU/ml; 53.5[+ or -]0.937 pg/ml, respectively) to almost normal levels (Figure 1 and 2).
Follow up of all rats was carried out by daily vaginal smear at 10:00-11:00AM after completion of CTX injections and for eight weeks following the intervention to check for the number of normal estrous cycles. In addition, fortnightly serum E2 and FSH estimation was carried out to assess the changes in ovarian function. Rats were sacrificed at the end of the study to detect the resurrection of ovarian activity (follicular growth and count).
On the 8th week of follow-up, the second and third groups showed that the mean FSH level was significantly lower in group II versus Group III (5.38 [+ or -] 0.31 vs. 19.11 [+ or -] 0.41mIU/ml; P<0.0001).
The mean E2 level showed a trend of rise in Group II than Groups III and IV from the second week of follow up onwards. This increase in mean E2 levels reached a highly significant difference by the eighth week (53.5[+ or -]0.93, 21.42[+ or -]0.48, and 19.42[+ or -]0.40 pg/ml, respectively).
It was evident that by the eighth week after induction of ovarian failure spontaneous recovery of the ovary did not occur for Groups III and IV as confirmed by mean FSH and E2 values that was not different statistically before and after intervention, where FSH was 19.10 [+ or -] 0.41 on the 8th week versus 7.98 [+ or -] 0.19 mIU/ml at the beginning of induction of ovarian failure for the 3rd group, P=0.1.
The mean level of FSH on the 8th week versus the start of CTX injections for group IV was 8.76 [+ or -] 0.22 vs. 7.88 [+ or -] 0.16mIU/ml, respectively; P=0.1. The same results were also obtained for the mean E2 level, where it was 21.42[+ or -]0.48 vs. 31.1[+ or -]0.54 pg/ml; P=0.03, respectively for Group III and 19.42[+ or -]0.40 vs. 30.90[+ or -]0.57 pg/ml; P=0.04, respectively in group IV by the 8th week of follow up versus the day following last dose of CTX.
These hormonal findings were confirmed by histological and cytological vaginal smear results (Figures III- VII). The histological findings of the ovaries of the sacrificed rats at the beginning of the trial, after completion of induction of failure, and at the end of the 8th week of the trial show that the ovaries of the rats treated with CTX exhibited cystic dilated follicles and some destructive and degenerative follicles. While, the ovaries of rats that received stem cells (Group II) appeared more or less as normal ovarian tissue with evidence of follicles and even corpus luteum by the end of eight weeks.
Also, our findings of the vaginal smears obtained from group II showed pro-estrous smears which were characterized by rounded, usually nucleated, epithelial cells, generally in low to moderate numbers and even some epithelial cells show early stages of cornification. Both these histological and cytological features were not detected in the groups that received normal saline (Group III) or the group that received nothing (Group IV).
In addition, PCR examination (Figures VIII- IX) of sry gene expression in the ovarian tissues obtained from Group II presented further evidence of stem cell incorporation into the ovarian structure. Such gene expression was not seen in any rat ovary from the other groups
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Basic research on how the ovary interacts with the immune system, particularly in the early stages of oogenesis, how the oocyte and follicle interacts in fetal life, and how follicles might be protected from damage in the case of immune self-attack are all fruitful avenues of clinically applicable work that may lead to treatments for the most vexing reproductive disorders. Furthermore, the current concept that the ovary has a static ovarian reserve is entirely at odds with germ cell dynamics in its counterpart, the testis.
The use of stem cells as a source of gametes also emerges as a hope in male and female cancer survivors. However, currently little information is available regarding the therapeutic potential of MSCs for chemotherapy-induced ovarian damage or women with idiopathic POF. (18) Moreover, the issue of stem cell research is still in its infancy and raises many ethical, moral and religious arguments. Most stem cell researches use cells obtained from adult tissue or umbilical cord blood sources that pose no moral problem. Nevertheless, the central setback regarding the human ovaries is that, the use of stem cells will propagate into future generations.
The present study found that MSCs implantation can ameliorate ovarian function in rats with ovarian failure. It was evident that secondary and antral follicles seem to be the primary targets of chemotherapy. Four weeks after transplantation, ovarian function was improved, as indicated by return of the estrous cycle, increased estradiol levels and follicle numbers, and decreased FSH levels. This was also shown in a study by a Chinese group but with the use of intra-peritoneal route. (19) Yet, the mechanisms behind the resurrection of ovarian activity and folliculogenesis is not fully elaborated, but the integration of MSCs into the tissue and replacement of damaged cells and the paracrine mediators secreted by MSCs might be involved in the repair process by preventing cell apoptosis and promoting functional recovery and induce DNA formation. (20) Speculatively, perhaps chemotherapy does not simply take away a number of eggs from a fixed ovarian reserve but interferes with the ability of the ovary to regenerate oocytes.
In several studies, it was established that MSCs secrete cytokines in vitro (21-22) including vascular endothelial growth factor, insulin-like growth factor -1 and human growth factor; in addition, MSCs inhibits chemotherapy-induced apoptosis of granulose cells in vitro (23) by up-regulating Bcl-2 protein and cytokines.
Bukovsky and his colleagues (24) have reported differentiation of distinct cell types from ovarian stem cells (OSC) and the production of new eggs in cultures derived from premenopausal and postmenopausal human ovaries. OSCs (Olfactory stem cells) are also capable of producing neural/neuronal cells in vitro after sequential stimulation with sex steroid combinations. Hence, OSC represent a unique type of totipotent adult stem cells, which could be utilized for autologous treatment of premature ovarian failure without the use of allogeneic embryonic stem cells or somatic cell nuclear transfer. This would overcome the ethical or moral questions regarding its use in POF. Nevertheless, its separation or isolation is not yet standardized in humans.
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Our work would substantiate the hypothesis of re-initiating new oocytes from ovarian surface epithelium (OSE) by observing the (sry) gene within the ovarian cells treated by male stem cells. This suggestion was not accepted by Lee and colleagues (17) who are skeptical about this transformation of OSE into new oocytes based on the assumption that the primordial follicles that seem to be morphologically normal under light microscopy may have ultra-structural damage and are eventually cleared from the ovary and the authors ruled out the possibility of an immediate germ cell bolus or a salvaging effect on pre-existing oocytes by the transplantations based on the fact that, even though the bone marrow-treated animals had severely diminished follicle reserve, the fertility rescuing effect was long lasting. They also found a small number of donor-originated oocytes, which were not mature enough to be ovulated, and none of the newborns were of donor origin. Thus, they hypothesized that the bone marrow treatment works through reinitiating new oocyte production in the host. They studied this in mice and have shown that bone marrow treatment generates donor-derived oocytes in CTX-treated recipients.
On the other hand, a subsequent report from an American group (25) claimed that ovulated eggs do not derive from bone marrow and that bone marrow-derived oocytes reported previously are misidentified immune cells. We believe that the return of ovarian activity was the result of incorporation of the stem cells within the ovarian tissue with transformation into the proposed cells. As proved by finding (Sry) gene (derived from male rats stem cells) within the ovarian tissue in female rats included in our study.
Conclusion: The ovary is more like its counterpart, the testis; it is not a static organ and continues to produce new germ cells into adult life. Overall, the present study confirmed that the ovarian damage induced by chemotherapy can be reverted by the use of MSCs therapy.
We proved that ovarian activity, both hormonally and folliculogenesis, were restored after incorporation of stem cells into the ovarian stroma, being the first to administrate stem cells intravenously for this purpose. Whether this animal observation can be extrapolated into human beings needs further research. If proven effective, this line of management could be used for autologous treatment of premature ovarian failure (spontaneous or chemotherapeutically induced).
Conflict of interest: None declared.
Corresponding author: Dr Osama Azmy, Professor, MD, FRCOG,DFFP Osama Azmy, 97 Montazah Street, Heliopolis 11351, Cairo, Egypt, e-mail: firstname.lastname@example.org
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 Asmaa Abbasy,  Osama Azmy,  Hazem Atta,  Ashraf Ali,  Laila Rashed,  Zakaria El-Khaiat,  Abd Elrazik Farrag,  Mamdouh Bibars,  Tamer Taha,  Walid El-Nattat,  Mohammed Talaat Abdel Aziz
 Obstetrics and Gynecology Department &  Medical Biochemistry and Molecular Biology Dept, Faculty of Medicine, Cairo University, Egypt
 Reproductive Health Research Dept,  Medical Biochemistry Dept,  Histology, Histochemistry, and Pathology Department,  Animal Reproduction and Artificial Insemination Department, National Research Centre, Cairo, Egypt
Table 1--The mean FSH [+ or -] SEM in the studied groups over the period of study After Before completion CTX of CTX Group I (Control) 2.86 [+ or -] 0.02 3.69 [+ or -] 0.11 Treated Group II 2.84 [+ or -] 0.03 8.39 [+ or -] 0.26 groups (CTX+MSCs) Group III 2.91 [+ or -] 0.06 7.98 [+ or -] 0.19 (CTX+Saline) Group IV 2.87 [+ or -] 0.04 7.88 [+ or -] 0.16 (CTX only) 2 Wk After 4 Wk After completion completion of CTX of CTX Group I (Control) 3.92 [+ or -] 0.07 4.02 [+ or -] 0.07 Treated Group II 8.27 [+ or -] 0.23 7.76 [+ or -] 0.24 groups (CTX+MSCs) Group III 11.35 [+ or -] 0.28 14.86 [+ or -] 0.38 (CTX+Saline) Group IV 10.42 [+ or -] 0.43 15.28 [+ or -] 0.35 (CTX only) 6 Wk After 8 Wk After completion completion of CTX of CTX Group I (Control) 3.89 [+ or -] 0.05 3.60 [+ or -] 0.08 Treated Group II 7.16 [+ or -] 0.24 5.38 [+ or -] 0.31 groups (CTX+MSCs) Group III 17.75 [+ or -] 0.35 19.10 [+ or -] 0.41 (CTX+Saline) Group IV 18.34 [+ or -] 0.29 18.76 [+ or -] 0.22 (CTX only) Table 2--The mean E2 [+ or -] SEM in the studied groups over the period of study After Before completion CTX of CTX Group I (Control) 63.11 [+ or -] 1.72 61.60 [+ or -] 1.42 Treated Group II 63.60 [+ or -] 0.99 32.38 [+ or -] 0.51 groups (CTX+MSCs) Group III 62.20 [+ or -] 0.95 31.10 [+ or -] 0.54 (CTX+Saline) Group IV 64.26 [+ or -] 1.27 30.90 [+ or -] 0.57 (CTX only) 2 Wk After 4 Wk After completion completion of CTX of CTX Group I (Control) 60.51 [+ or -] 1.48 60.60 [+ or -] 1.30 Treated Group II 35.30 [+ or -] 0.48 38.22 [+ or -] 0.62 groups (CTX+MSCs) Group III 24.06 [+ or -] 0.44 23.21 [+ or -] 0.45 (CTX+Saline) Group IV 24.63 [+ or -] 0.45 23.36 [+ or -] 0.23 (CTX only) 6 Wk After 8 Wk After completion completion of CTX of CTX Group I (Control) 60.67 [+ or -] 1.31 69.71 [+ or -] 1.26 Treated Group II 47.50 [+ or -] 0.67 53.50 [+ or -] 0.93 groups (CTX+MSCs) Group III 22.05 [+ or -] 0.51 21.42 [+ or -] 0.48 (CTX+Saline) Group IV 21.64 [+ or -] 0.40 19.42 [+ or -] 0.40 (CTX only)