Background
Ovarian tissue cryopreservation is an experimental fertility preservation method and the transplantation techniques are still evolving.
Objective
We attempted to improve the technique with the utility of a human decellularized extracellular tissue matrix (ECTM) scaffold, robot-assisted minimally invasive surgery, and perioperative pharmacological support.
Study Design
We prospectively studied 2 subjects with hemophagocytic lymphohistiocytosis (patient A) and non-Hodgkin lymphoma (patient B) who underwent ovarian tissue cryopreservation at the age of 23 years, before receiving preconditioning chemotherapy for hematopoietic stem cell transplantation. Both experienced ovarian failure postchemotherapy and we transplanted ovarian cortical tissues to the contralateral menopausal ovary 7 and 12 years later, using a human ECTM scaffold and robotic assistance. The ECTM scaffold tissue compatibility was shown in preclinical studies. Patients also received estrogen supplementation and baby aspirin preoperatively to aid in the revascularization process.
Results
Ovarian follicle development was observed approximately 10 (patient A) and 8 (patient B) weeks after ovarian tissue transplantation. Following 8 and 7 cycles of in vitro fertilization, 9 and 10 day-3 embryos were cryopreserved (patients A and B, respectively). While the baseline follicle-stimulating hormone (range 3.6-15.4 mIU/mL) levels near normalized by 7 months and remained steady postovarian transplantation in patient A, patient B showed improved but elevated follicle-stimulating hormone levels throughout (range 21-31 mIU/mL). Highest follicle yield was achieved 14 (8 follicles; patient A) and 11 (6 follicles; patient B) months postintervention. Patient A experienced a chemical pregnancy after the third frozen embryo transfer attempt. She then conceived following her first fresh in vitro fertilization embryo transfer and the pregnancy is currently ongoing. Patient B conceived after the first frozen embryo transfer attempt and delivered a healthy girl at term.
Conclusion
We report the first pregnancies after the minimally invasive transplantation of previously cryopreserved ovarian tissue with an ECTM scaffold. This approach seems to be associated with steady ovarian function after a follow-up of up to 2 years.
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Introduction
Ovarian cryopreservation is one of the key strategies in fertility preservation. Utilizing the previously accumulated knowledge from animal and human ovarian xenografting studies, we performed the first autologous ovarian tissue transplantation (OTT) case with frozen-thawed tissue in 1999. Although the patient did not desire pregnancy, she demonstrated ovarian follicle development 8 weeks after the transplant with documented function for up to 9 months. The live births were to follow several years later as reported by numerous investigators. In addition, we reported heterotopic OTT techniques where the tissues were grafted subcutaneously under the forearm or abdominal skin. The latter resulted in oocyte retrievals and embryo development.
With the current techniques, akin to skin grafting, one has to rely on the natural process of revascularization from the recipient site. Because it takes up to 10 days for new human ovarian microvessels to reach full maturity, the graft suffers an initial ischemic injury. This initial ischemic injury has been shown to result in the loss of nearly two thirds of all primordial follicles in ovarian xenograft models. This inefficiency largely explains the unpredictable longevity of ovarian transplants. The length of ovarian function has been reported to range from 1 to >7 years with an average of 4-5 years in successful orthotopic and heterotopic ovarian transplants with frozen-banked ovarian tissue.
In addition, issues with oocyte quality have been cited by some after in vitro fertilization (IVF); one possible explanation being the restricted blood flow. The baseline follicle-stimulating hormone (FSH) levels remain high and anti-müllerian hormone (AMH) levels tend to be low in the majority of ovarian transplants with cryopreserved tissue, possibly reflecting the restriction in blood flow. Therefore, there is convincing evidence to support the view that vascularization issues curb OTT success both acutely in the form of follicle loss and chronically with limited microvascular flow.
AlloDerm (LifeCell Corp, Branchburg, NJ) is a decellularized human extracellular tissue matrix (ECTM) generated from cadaver skin. It has been used in cosmetic surgery, breast reconstruction, and dentistry and other surgical reconstructive fields to augment tissue grafts and aid in revascularization. In this study we hypothesized that the cryopreservation of ovarian tissue may preserve fertility. We also hypothesized that the use of the ECTM with robotic surgical assistance may improve outcomes, presumably by aiding the revascularization process.
Materials and Methods
This translational work represents the outcomes ovarian cryopreservation and transplantation research protocol after up to 14-year follow-up of 2 subjects who consecutively underwent grafting. The study was approved by the institutional review board and the preclinical animal studies were also approved by the Institutional Animal Care and Use Committee at New York Medical College.
Patient A was diagnosed with familial hemophagocytic lymphohistiocytosis in April 2006, at the age of 23 years. Shortly before the preconditioning chemotherapy (thiotepa and fludarabine) and total body irradiation (1375 cGY) for hematopoietic stem cell transplant, we performed a laparoscopic oophorectomy. Ovarian cortical strips were cryopreserved with a slow freeze protocol. In April 2013, her blood work was consistent with menopausal state (FSH 108.7 mIU/mL; luteinizing hormone [LH] 61.4 mIU/mL; estradiol [E 2 ] <6 pg/mL; AMH <0.16 ng/mL) and a transvaginal ultrasound showed that the remaining ovary and the endometrium to be atrophic.
Patient B was diagnosed with non-Hodgkin lymphoma at the age of 17 years and completed a chemotherapy regimen including cyclophosphamide (600 mg/m 2 × 8 courses every 3 weeks), prednisone, procarbazine, doxorubicin, bleomycin, and vinblastine in October 1996. She experienced mediastinal recurrence at the age of 23 years. We performed ovarian cryopreservation with a protocol identical to patient A, shortly before receiving the highly gonadotoxic regimen of ifosfamide, carboplatin, etoposide, and dexamethasone prior to hematopoietic stem cell transplant. In July 2013, her FSH and LH levels were found to be 54 and 70 mIU/mL, and 47 and 33 mIU/mL 1 month apart, respectively. Her AMH was <0.16 ng/mL with ultrasound examination showing no obvious healthy follicles, confirming the diagnosis of ovarian failure.
Follicle density assessment
In patient A, because the diagnosis was noncancer and an ovarian sample to rule out metastasis was not needed at the time of ovarian tissue cryopreservation we could not assess precryopreservation follicle density. However, 1 vial of tissue was thawed and the follicle density was assessed before the OTT. This revealed a mean follicle density of 1.66 ± 0.37 follicles/mm 2 . Based on these results, and after discussion with the couple, we empirically decided to thaw 5 of 10 vials, containing 10 pieces of ovarian tissue for transplantation.
Likewise, in patient B, 1 vial of tissue was thawed prior to OTT, and revealed a mean follicle density of 0.62 ± 0.32 follicles/mm 2 . Although patient B was of similar age to patient A at the time of ovarian tissue cryopreservation, this density was significantly lower than that of with patient A ( P < .05). Furthermore, when compared to patient’s precryopreservation follicle density of 1.4 follicles/mm 2 , >50% of follicles appeared to have been lost during freezing and thawing. Based on these results and following a discussion with the couple, we empirically decided to thaw and transplant 6 of 12 vials, containing 12 pieces of ovarian tissue.
Preoperative preparation
Before the procedure, both patients underwent a hysterosonogram and the partners were evaluated with a semen analysis. In addition, patient A also underwent a pelvic magnetic resonance imaging to rule out any obvious total body irradiation–induced uterine damage. Ten weeks prior to the procedures, both patients received transdermal 0.1 mg E 2 (Climara; Bayer Healthcare Pharmaceuticals Inc, Whippany, NJ) weekly and vaginal progesterone 100 mg (Prometrium; Schering-Plough, Kenilworth, NJ) nightly with a 2-week-on/2-week-off regimen. This regimen was continued after the transplant until a sign of ovarian function was seen. Hormone replacement was given as there is some evidence from animal studies that this may improve ovarian vascularization. Again, with the aim of enhancing revascularization, both patients were also given daily baby aspirin 81 mg (Bayer Healthcare Pharmaceuticals Inc) for 7 days, which was discontinued 2 days before the surgery.
Preclinical evaluation of ECTM
This ECTM has been used in the surgical field but there is no description of its use in reproductive surgery. To ensure its compatibility with ovarian tissues, we performed a series of preclinical evaluations. First we evaluated various thicknesses (thin, medium, full) of the ECTM in thawing media ( Figure 1 , A) as well as simulating its use with ovarian cortical pieces from organ donor cadavers to determine the best thickness for handling. We found the medium-thickness ECTM to be sturdy yet malleable and hence next tested it in a xenograft model. We subcutaneously xenografted 4- × 4-mm ovarian cortical pieces together with ECTM to immunodeficient mice ( Figure 1 , B), as we previously described. After 10 days of xenografting, the tissues were evaluated. We found that ovarian stroma had integrated into ECTM, without any pathological changes ( Figure 1 , C and D).
Next we cultured mouse oocytes (N = 30/group) with or without ECTM for 16 hours and assessed survival. We found that there was no difference in oocyte survival when ECTM was used, compared to controls (88.1% vs 81.6%, P = .39). These findings provided assurance that ECTM was compatible with ovarian tissues.
Ovarian transplant technique
Robotically assisted OTT procedures were performed in July 2013 on patient A at the age of 30 years (7 years after ovarian cryopreservation), and on patient B in October 2013 at the age of 35 years (12 years after cryopreservation). The OTT technique is described in Supplement 1 and illustrated in Video S1 .
Statistical methods
Statistical analyses were performed with software (SPSS 15 for Windows; IBM Corp, Armonk, NY) using Student t test (follicle density comparisons) or Fisher exact test (survival rates of mouse oocytes). A P value of ≤.05 was considered statistically significant.
Materials and Methods
This translational work represents the outcomes ovarian cryopreservation and transplantation research protocol after up to 14-year follow-up of 2 subjects who consecutively underwent grafting. The study was approved by the institutional review board and the preclinical animal studies were also approved by the Institutional Animal Care and Use Committee at New York Medical College.
Patient A was diagnosed with familial hemophagocytic lymphohistiocytosis in April 2006, at the age of 23 years. Shortly before the preconditioning chemotherapy (thiotepa and fludarabine) and total body irradiation (1375 cGY) for hematopoietic stem cell transplant, we performed a laparoscopic oophorectomy. Ovarian cortical strips were cryopreserved with a slow freeze protocol. In April 2013, her blood work was consistent with menopausal state (FSH 108.7 mIU/mL; luteinizing hormone [LH] 61.4 mIU/mL; estradiol [E 2 ] <6 pg/mL; AMH <0.16 ng/mL) and a transvaginal ultrasound showed that the remaining ovary and the endometrium to be atrophic.
Patient B was diagnosed with non-Hodgkin lymphoma at the age of 17 years and completed a chemotherapy regimen including cyclophosphamide (600 mg/m 2 × 8 courses every 3 weeks), prednisone, procarbazine, doxorubicin, bleomycin, and vinblastine in October 1996. She experienced mediastinal recurrence at the age of 23 years. We performed ovarian cryopreservation with a protocol identical to patient A, shortly before receiving the highly gonadotoxic regimen of ifosfamide, carboplatin, etoposide, and dexamethasone prior to hematopoietic stem cell transplant. In July 2013, her FSH and LH levels were found to be 54 and 70 mIU/mL, and 47 and 33 mIU/mL 1 month apart, respectively. Her AMH was <0.16 ng/mL with ultrasound examination showing no obvious healthy follicles, confirming the diagnosis of ovarian failure.
Follicle density assessment
In patient A, because the diagnosis was noncancer and an ovarian sample to rule out metastasis was not needed at the time of ovarian tissue cryopreservation we could not assess precryopreservation follicle density. However, 1 vial of tissue was thawed and the follicle density was assessed before the OTT. This revealed a mean follicle density of 1.66 ± 0.37 follicles/mm 2 . Based on these results, and after discussion with the couple, we empirically decided to thaw 5 of 10 vials, containing 10 pieces of ovarian tissue for transplantation.
Likewise, in patient B, 1 vial of tissue was thawed prior to OTT, and revealed a mean follicle density of 0.62 ± 0.32 follicles/mm 2 . Although patient B was of similar age to patient A at the time of ovarian tissue cryopreservation, this density was significantly lower than that of with patient A ( P < .05). Furthermore, when compared to patient’s precryopreservation follicle density of 1.4 follicles/mm 2 , >50% of follicles appeared to have been lost during freezing and thawing. Based on these results and following a discussion with the couple, we empirically decided to thaw and transplant 6 of 12 vials, containing 12 pieces of ovarian tissue.
Preoperative preparation
Before the procedure, both patients underwent a hysterosonogram and the partners were evaluated with a semen analysis. In addition, patient A also underwent a pelvic magnetic resonance imaging to rule out any obvious total body irradiation–induced uterine damage. Ten weeks prior to the procedures, both patients received transdermal 0.1 mg E 2 (Climara; Bayer Healthcare Pharmaceuticals Inc, Whippany, NJ) weekly and vaginal progesterone 100 mg (Prometrium; Schering-Plough, Kenilworth, NJ) nightly with a 2-week-on/2-week-off regimen. This regimen was continued after the transplant until a sign of ovarian function was seen. Hormone replacement was given as there is some evidence from animal studies that this may improve ovarian vascularization. Again, with the aim of enhancing revascularization, both patients were also given daily baby aspirin 81 mg (Bayer Healthcare Pharmaceuticals Inc) for 7 days, which was discontinued 2 days before the surgery.
Preclinical evaluation of ECTM
This ECTM has been used in the surgical field but there is no description of its use in reproductive surgery. To ensure its compatibility with ovarian tissues, we performed a series of preclinical evaluations. First we evaluated various thicknesses (thin, medium, full) of the ECTM in thawing media ( Figure 1 , A) as well as simulating its use with ovarian cortical pieces from organ donor cadavers to determine the best thickness for handling. We found the medium-thickness ECTM to be sturdy yet malleable and hence next tested it in a xenograft model. We subcutaneously xenografted 4- × 4-mm ovarian cortical pieces together with ECTM to immunodeficient mice ( Figure 1 , B), as we previously described. After 10 days of xenografting, the tissues were evaluated. We found that ovarian stroma had integrated into ECTM, without any pathological changes ( Figure 1 , C and D).
Next we cultured mouse oocytes (N = 30/group) with or without ECTM for 16 hours and assessed survival. We found that there was no difference in oocyte survival when ECTM was used, compared to controls (88.1% vs 81.6%, P = .39). These findings provided assurance that ECTM was compatible with ovarian tissues.
Ovarian transplant technique
Robotically assisted OTT procedures were performed in July 2013 on patient A at the age of 30 years (7 years after ovarian cryopreservation), and on patient B in October 2013 at the age of 35 years (12 years after cryopreservation). The OTT technique is described in Supplement 1 and illustrated in Video S1 .
Statistical methods
Statistical analyses were performed with software (SPSS 15 for Windows; IBM Corp, Armonk, NY) using Student t test (follicle density comparisons) or Fisher exact test (survival rates of mouse oocytes). A P value of ≤.05 was considered statistically significant.
Results
Patient A
The first sign of ovarian activity was detected approximately 10 weeks after OTT in October 2013, by the demonstration of a 14.5-mm follicle on a transvaginal ultrasound exam. The hormone replacement was then discontinued and the patient resumed cyclical menstruation ( Figure 2 , A).
