Autologous in vitro cultured vaginal tissue for vaginoplasty in women with...

Accepted Manuscript

Autologous in vitro cultured vaginal tissue for vaginoplasty in women with Mayer–Von-Rokitansky–Küster–Hauser syndrome: anatomical and functional results

Pierluigi Benedetti Panici, M.D. Diana Maffucci, M.D. Simona Ceccarelli, PhD EnricaVescarelli, Giorgia Perniola, M.D. Ludovico Muzii, M.D. Cinzia Marchese, PhD

PII: S1553-4650(14)01421-6

DOI: 10.1016/j.jmig.2014.09.012

Reference: JMIG 2393

To appear in: The Journal of Minimally Invasive Gynecology

Received Date: 9 June 2014

Revised Date: 25 September 2014

Accepted Date: 28 September 2014

Please cite this article as: Benedetti Panici P, Maffucci D, Ceccarelli S, Vescarelli E, Perniola G, MuziiL, Marchese C, Autologous in vitro cultured vaginal tissue for vaginoplasty in women with Mayer–Von-Rokitansky–Küster–Hauser syndrome: anatomical and functional results, The Journal of MinimallyInvasive Gynecology (2014), doi: 10.1016/j.jmig.2014.09.012.

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http://dx.doi.org/10.1016/j.jmig.2014.09.012

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TITLE

Autologous in vitro cultured vaginal tissue for vaginoplasty in women with Mayer–Von-

Rokitansky–Küster–Hauser syndrome: anatomical and functional results.

Pierluigi BENEDETTI PANICI M.D.1, Diana MAFFUCCI M.D.1*, Simona CECCARELLI PhD2,

Enrica VESCARELLI2, Giorgia PERNIOLA M.D.1, Ludovico MUZII M.D.1, Cinzia MARCHESE

PhD2.

1 Department of Gynecologic-Obstetrical and Urologic Sciences .“Sapienza” University of Rome.

Viale del Policlinico, 155 – 00161, Rome. Italy

2 Department of Experimental Medicine - Unit of Regenerative Medicine. “Sapienza” University of

Rome. Viale Regina Elena, 324 – 00161, Rome. Italy

The Authors report no conflict of interest

The authors have no disclosure to report

*Corresponding author: Diana Maffucci, M.D.

E-mail: [email protected]

Business telephone number: + 393395018918

Fax: + 39064454820

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PRÉCIS

This study presents the procedure and the results of a technique in which in vitro autologous cell

cultures were used for the canal lining in patients with Mayer–von-Rokitansky–Küster–Hauser

syndrome (MRKHS)

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STRUCTURED ABSTRACT

STUDY OBJECTIVE: To present the procedure and the results of a technique in which in vitro

autologous cell cultures were used for the canal lining in patients with Mayer–von-Rokitansky–

Küster–Hauser syndrome (MRKHS) subjected to vaginoplasty with a modified Abbè–McIndoe

technique. MRKHS is a rare anomaly characterized by vaginal agenesis with variable Müllerian

duct abnormalities. The Abbè-McIndoe procedure is one of the most frequent surgical treatments

adopted in these women. In the last decades several modifications have been introduced by different

authors, mostly changing the lining material, but no consensus has been reached on what material

should be used for the neovagina canal wall lining.

DESIGN: Pilot study.

DESIGN CLASSIFICATION: II-1 according to the Canadian Task Force Classification of Study

Designs.

SETTING: Policlinico Umberto I, “Sapienza” University of Rome.

PATIENTS: A consecutive series of 23 women with MRKHS underwent neovaginoplasty with

autologous vaginal tissue as graft material, between 2006 and 2013.

INTERVENTIONS: Each patient with MRKHS was subjected to a full-thickness mucosal biopsy

from the vaginal vestibule. Following enzymatic dissociation, cells were inoculated onto collagen

IV-coated plates and cultured for 2-3 weeks. The patients were subjected to a vaginoplasty with a

modified Abbè–McIndoe technique with autologous in vitro cultured vaginal tissue. Patients

underwent clinical follow-up visits at 1, 3, 6 and 12 months after surgery and every year thereafter.

Anatomical, functional and sexual results were assessed.

MEASUREMENTS AND MAIN RESULTS: In all cases the vagina appeared normal in length

and depth. A vaginal cytology and a vaginal biopsy obtained at 3 month follow-up visit revealed

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physiological vaginal tissue. All 23 patients completed the Female Sexual Function Index (FSFI)

questionnaire at 12 months after surgery. The results showed a total score of 27.2.

This results demonstrate a satisfactory quality of sexual life.

CONCLUSION: The modified Abbè–McIndoe technique with autologous vaginal tissue appears

safe and feasible. This technique allows normal and satisfying sexual intercourse. Larger series with

longer follow-up will be necessary to confirm if this technique represents the ideal procedure for

vaginal agenesis.

Key Words: Abbè-McIndoe, Autologous Tissue, Mayer-Rokitansky-Küster-Hauser syndrome,

vaginal agenesis, vaginoplasty.

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TEXT

Introduction

The estimated incidence of Mayer–von-Rokitansky–Küster–Hauser syndrome (MRKHS) is 1:

5.000 live-born females (1-2). The aetiology of MRKHS is considered multifactorial (3).

Women affected by MRKHS have normal female phenotype and karyotype (46, XX).

Ovarian function is usually preserved. Most women have a complete uterine agenesis or a non-

functional rudimental uterus. Patients suffering from MRKHS usually maintain a normal vaginal

vestibule and sometimes a vaginal dimple.

The symptom that usually leads to diagnosis is primary amenorrhea. In patients with MRKHS,

vaginal aplasia/hypoplasia is frequently associated with other congenital abnormalities.

Approximately a third of these women have renal system abnormalities and skeletal malformations

and a minority of the cases may have cardiovascular anomaly. Müllerian remnants can be

frequently observed in these patients (4).

The objective of the treatment of vaginal agenesis is to allow women to have a satisfactory sexual

life. The ideal treatment should aim to achieve an anatomically and functionally normal vagina.

Ideally, the procedure should be able to be permanent, require minimal use of dilators and be non or

minimally invasive without leaving visible scars.

Several methods have been suggested for the treatment of vaginal agenesis. Currently, the

American College of Obstetrics and Gynecology (ACOG) suggests a conservative approach (5-6) as

first line treatment and reserve surgery as a second line treatment in patients who failed progressive

dilation or who are not compliant (2).

Several surgical techniques have been developed for the treatment of vaginal agenesis.

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Recently, the interest of most gynaecologists has been directed towards minimally invasive

techniques (7). The most widely adopted minimally invasive methods are the laparoscopic

Vecchietti’s technique and the Abbé-McIndoe procedure.

The Vecchietti’s technique (8) traditional or laparoscopically-modified (9-10), commonly used in

European countries, is carried out using an acrylic olive placed in the vaginal dimple with two

threads that are passed through the abdominal cavity which are attached to a traction device placed

on the sovrapubic region. The threads are subjected to a progressive traction until the vaginal

dimple is elongated to the desired length; so the patients have to adjust the tension every 48 hours.

The critical point of this procedure is the prolonged time required, usually between 6 and 9 days,

necessary to obtain an adequate vaginal length. The postoperative period is uncomfortable and

furthermore an excessive traction may cause necrosis of epithelium, whereas limited traction would

not allow lengthening of the neovagina.

The Abbè-McIndoe technique (11) is a minimally invasive procedure that is widely adopted for the

treatment of vaginal agenesis. This technique consists in the creation of a canal in between the

bladder and the rectum, which is covered with a skin graft. Several modifications of this original

technique have been attempted, mostly changing the different tissue adopted for the canal lining.

Here we present our continued experience with this novel treatment initially described by our group

in a case report (12) in which in vitro autologous vaginal cell cultures obtained from biopsies from

the vaginal vestibule were used for the epithelialization of the neovaginal walls.

This prospective pilot study was carried out on 23 women with MRKH syndrome who were treated

in our Department between 2006 and 2013.

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Methods

IRB approval was obtained.

Preoperative workup

Between 2006 and 2013, 23 caucasian women with MRKH syndrome were addressed to our

institution. All patients exhibited primary amenorrhoea, normal secondary sex characteristics, and a

vaginal dimple without vaginal orifice.

All women underwent a pre-operative work-up, which included chromosome analysis, abdomino-

pelvic MRI, echocardiogram, ECG and chest X-ray.

Patients were counseled, before surgery, regarding the non-surgical technique (Frank’s method) and

alternative surgical techniques, as well as the possible complications of the modified Abbè–

McIndoe technique with autologous in vitro cultured vaginal tissue.

The technique has been described by our groups previously (12).

Surgical technique

The technique consists in a two-step procedure. The patient is subjected to a preliminary full

thickness biopsy from the vaginal vestibule of approximately 1cm2 (Figure 1).

The fresh biopsy is transferred to the Department of Experimental Medicine for cell extraction and

tissue culture. Following enzymatic dissociation, the keratinocyte suspension is inoculated onto five

collagen IV (10 mg/ml)-coated culture plates, as previously reported (12). Cells are seeded at cell

density of 2.5 X 105 cells/plate and maintained in Keratinocyte Basal Medium (KBM, Lonza

Milano S.r.l., Milano, Italy) supplemented with Keratinocyte Growth Medium (KGM) single quotes

(Lonza), with medium change twice a week. The size of the tissue culture is determined by the

number and size of the plates adopted.

After 1-2 weeks of culture, cells reach 70–80% of confluence, being cultured for additional 8 days

in order to obtain a fully differentiated mucosal tissue. Autologous reconstructed vaginal tissues are

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then harvested from the culture plates by incubation with dispase II (2.5 mg/ml), washed in PBS,

mounted onto 2 mg/10 cm² hyaluronic acid embedded gauze to maintain the orientation of the

mucosal tissue and transferred to the operative theatre, taking care that the basal epithelial cell layer

faced away from the gauze (Figure 2: 2a-2b-2c).

The time interval between the biopsy and the development of a fully differentiated mucosal tissue is

approximately 2-3 weeks.

Before the second-step surgery, the patients are treated with antibiotics prophylaxis and a bowel

enema. In the operative room, the patients are placed in a lithotomic position with a Foley catheter.

The vaginal canal is developed as described in the Abbè-McIndoe technique: a midline incision at

the vaginal introitus is made, and a 10 cm canal is made between the bladder and the rectum using a

blunt digital and scissor dissection reaching the pouch of Douglas.

Complete haemostasis is obtained with electrocautery (Figure 3: 3a-3b-3c-3d). The tunnel is

covered with the gauze with the cell stratum facing the canal walls. The gauzes are kept in place by

a 2 cm in diameter and 12 cm in length vaginal mould (Figure 4: 4a-4b-4c). The mould is fixed to

the perineum. No stitches are used to keep the gauze in place.

The gauzes, the Foley catheter and the mould are kept in place for 5 post-operative days. At this

point, the mould is removed, the vagina is rinsed and the percentage of take is evaluated. The Foley

catheter was kept in place until the patients started using the dilators autonomously.

The patients are advised to wear the vaginal mould day and night during the initial 6 weeks post-

operative period, after which the mould is kept at night until regular sexual intercourse is

commenced.

Patients underwent clinical follow-up visits at 1, 3, 6 and 12 months after surgery and every year

thereafter. Clinical follow-up included vaginal examination and vaginoscopy.

Both vaginal cytology and vaginal biopsy were obtained at the 3 months follow-up visit.

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Functional results were assessed by using Female Sexual Function Index (FSFI) questionnaire , at

12 months after surgery.

The highest total FSFI score that may be obtained is 36. On the basis of literature data, 30 is the

mean score in the normal population, so a functional result can be considered as good when the total

score is between 30 and 36, satisfactory when between 23 and 29, poor when is 23 or less (13).

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Results

The operation was performed successfully in all 23 patients. Patients characteristics and primary

results are presented in Table I. The mean age of patients at surgery was 21.7 years-old (range 17-

49 years-old). The measure of the vaginal biopsy ranged from 0.5 to 2 cm² (mean 1.4 cm²).

The mean operative time was 21.5 minutes (range 18- 25 min) and mean blood loss was 94 ml

(range 80 -110 ml). The time interval between the biopsy and the development of the fully

differentiated mucosal tissue ranged from 15 to 27 days (mean time 20.4 days).

The vaginal tissue percentage of take was between 70 and 100% (mean percentage 83%).

The evaluation of neovagina with gynecologic examination, carried out after 5 days from surgery,

demonstrated that the vaginal tissue covered the 70-100 % of the vaginal wall, whereas the

remainder of neovagina appeared covered by granulation tissue, which was successively replaced

by vaginal tissue up to 2 weeks after.

Post-operatively, all the patients were placed on a liquid diet until day 5. The Foley catheter was

kept in place until the patients started using the dilators autonomously.

All patients urinated smoothly after bladder training. The hospitalization stay ranged from 2 to 16

days. Patients started using the dilators autonomously after a mean post-operative time of 12 days

(range 9-16 days).

At time of discharge patients were suggested to use liquid lubricants in order to improve the

compliance to the use of dilators.

One patient suffered from postoperative bleeding that was treated with vaginal packing and medical

therapy administering intravenously tranexamic acid.

One patient suffered from vaginal spotting at three month from surgery for granulomatous tissue.

Anatomic and functional success was achieved in all patients. Sexual activity was commenced from

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1 to 4 month after surgery. Patients started a regular sexual activity after a mean post-operative time

of 7 weeks (range 4-16 weeks).

Mean vaginal length was 8 cm ranging from 6 cm to 10 cm, and the width was about two fingers in

all patients. The vaginal length and depth at 12 months of follow-up did not differ, compared with

that at 6 post-operative months.

There was no exudate, and the vaginal tunnel was covered with a pink smooth lining.

The vaginal cytology and the vaginal biopsy of the neovagina obtained at 3 month follow-up visit

did not reveal atrophy and showed a normal epithelium (Figure 5).

All 23 patients completed the FSFI questionnaire at 12 months after surgery. The results showed a

total score of 27.2. This results demonstrate a satisfactory quality of sexual life. This finding

demonstrates that vaginoplasty with autologous in vitro cultured vaginal tissue permits normal and

satisfying sexual intercourse.

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Discussion

Various treatments have been proposed in the last century for vaginal agenesis.

Every technique shows advantages and disadvantages, so the ideal treatment of MRKHS remains a

matter of debate. Invasive reconstruction of the vaginal canal can be carried out using bowel

segments (14-15) or skin flaps (16).

In recent years, several Authors have advocated the use of non-invasive methods or minimally

invasive procedures as the ideal strategies to create a functional vagina.

Currently, the conservative approach such as the Frank or the Ingram method (5-6) represents the

standard first line treatments in motivated patients with favourable anatomic conditions (2), but the

long duration of treatment often leads to prefer surgery.

The most widely adopted minimally invasive methods are the laparoscopic Vecchietti’s technique

(8) and the Abbé-McIndoe procedure (11).

Since the first description of Abbè, several modifications of the original procedure have been

proposed, the most relevant ones regarding the tissue for the canal lining.

The original technique has demonstrated to be highly feasible and yield high success rates but carry

the inherited disadvantage of leaving a scar in the donor site.

Modified techniques can obviate several of these inconveniences and various modifications have

been proposed. These include the use of the peritoneum (17-18), the amnion (19-20), allogeneic

epidermal sheets (21-22), Interceed (23) and autologous buccal mucosa (24).

The use of peritoneum has the disadvantage of being complex, requires the opening of the

peritoneal cavity and the retraction of the labia minor in the vagina has been reported (25).

Vaginoplasty with amniotic membranes is a rapid procedure and does not leave any scar.

Furthermore, membranes can be stored for several days, and this allows physicians to have time to

organize the transplant. Epithelialization has constantly been shown to be rapid.

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The use of allogeneic tissues such as amnion sheets carries the inherited disadvantage of allograft

rejection and risk of human-to-human transmission of infective disease. Lastly, amniotic

membranes have been shown to be easily contaminated before or after being transplanted.

Inert materials are inexpensive, readily available, sterile, and allow fast operative times with no

cutaneous scars. With these materials, epithelialization occurs either from metaplasia or from

keratinocyte colonization from the vaginal vestibule and therefore the interval between surgery and

the beginning of intercourse is relatively long.

Buccal mucosa appears as a promising lining material for the neovagina . Regarding vaginoplasty

performed with autologous buccal micromucosa, Li et al (26) recently reported the long-term

outcome of 38 patients subjected to this type of technique. They demonstrated that this technique is

an effective and feasible approach, but needs a long operative times and two operative teams: one

for oral mucosa graft harvesting and the other for reconstruction of the neovaginal cavity. Other

possible problems related to this method are morbidity connected with the removal of the mucosa

from the donor site and the relatively long time necessary to achieve a functional vagina.

Comparing to our technique, vaginoplasty with oral micromucosa showed a longer operative time

(86.4 minutes vs 21.5 minutes) but similar FSFI score (28.8 vs 27.2).

The idea of regenerate tissue through a process of cultured cells is an attractive paradigm to be

explored. A pilot cohort study assessing the use of engineered vaginal organs in four patients with

MRKHS was recently published (27).

The use of in vitro cultured vaginal mucosa is in its early infancy and more experience needs to be

acquired before drawing definite conclusions.

In our patients no use of resorbable scaffolds, growth factors and peptides has been exploited for

controller delivery and release of the new mucosal tissue. Since these reconstructed mucosal

equivalents are fragile and difficult to handle in the clinical setting, only epithelial/mucosal sheets

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covered by a gauze of degradable hyaluronic acid, that confers both mechanical stability and high

biocompatibility and allowed the orientation of the epithelial layer has been used.

The technique appears to carry several advantages. The graft is autologous and orthotopic with no

risk of rejection or donor-derived infections.

The procedure is fast and leaves no visible scars. Epithelialization is rapid since there is no need to

“mesh” the material, but there is the possibility of having enough tissue to cover the entire canal

surface.

Furthermore, after removing the gauze and the mould, adopted in order to maintain in place and to

increase the pressure on the gauzes with vaginal tissue, the epithelialization could be seen through

the entire neovaginal wall and this suggest that epithelialization occurred from the transplanted

tissue and not as with other techniques from the vaginal vestibule.

As expected, the initial percentage of epithelialization of neovagina was variable between

individual patients. We didn’t investigate the possible reasons of this variability. Future

investigation will clarify the role of age, cell characteristics, medications and/or environmental

factors in determining the lining of neovagina.

In addition, women’s perception to have the vagina lined by physiological vaginal tissue is likely to

play an important psychological role in the achievement of a normal sexual life.

Moreover, we recently published a detailed characterization of cultured vaginal mucosa cells,

demonstrating their epithelial features and the presence of mucified cells interspersed among cell

cultures, which might contribute to maintain a normal glandular function of the neovagina (28).

Disadvantages include that the cell-based therapies can only be carried out in culture conditions

compliant with Good Manufacturing Practice (GMP) which require centres who have dedicated

tissue culture laboratories (such as referral centres or hospitals with burn units). Moreover, the fact

that the procedure is carried out in two steps means that the operative theatre organization need to

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be coordinated with the laboratories in order to program the surgical procedure when the tissue is

ready.

The quality of sexual life is the main objective of the treatment. The FSFI is the questionnaire used

to evaluate the satisfactory or unsatisfactory sexual intercourse after the surgery. The results

demonstrate a satisfactory quality of sexual life.

Future studies should also consider to extend the use of autologous vaginal tissue for the

reconstruction of acquired defects of the vagina resulting from extirpative oncologic surgery of

cervical, vaginal or urinary tract malignancies, or from traumas or infections with soft tissue

necrosis and subsequent extensive debridement. Such defects are generally repaired or

reconstructed with fasciocutaneous and myocutaneous flaps, but in oncologic patients previously

treated with radiotherapy the surrounding tissues are often subjected to an increased risk of wound

healing complications. The use of autologous cultured vaginal tissue could prevent this problem

since vaginal biopsy could be obtained before the beginning of radiotherapy.

In conclusion, we demonstrated that autologous in vitro cultured vaginal tissue combines the best

material characteristics with important psychological implications. Larger series with longer follow

up will be required to confirm if this technique represents the ideal procedure for vaginal agenesis

and if it can be applied also for reconstruction of acquired vaginal defects.

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BioMed Res Int. 2014;2014:201518.

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TABLE

Table 1: Patients characteristics and primary results.

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FIGURE LEGENDS

Figure 1: Full-thickness mucosal biopsy obtained from the vaginal vestibule.

Figure 2 (2a-2b-2c): In vitro culture of vaginal mucosa cells (PAS staining, identifying mucinous

cells and phase contrast) and detached tissues with application of hyaluronic acid embedded gauzes.

Figure 3 (3a-3b-3c-3d): Modified Abbè–McIndoe vaginoplasty: a canal reaching the pouch of

Douglas was made between the bladder and the rectum using blunt digital and scissor dissection.

Figure 4 (4a-4b-4c): Modified Abbè–McIndoe vaginoplasty: the tunnel was covered with the

gauzes with the mounted cell stratum facing the canal walls.

Figure 5: Vaginoscopy carried out during a 3- months interval patient’s follow-up visit.

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CASE AGE Biopsy

specimen

size

(cm²)

Blood

loss

(ml)

Operative

time

(min.)

Tissue

take

(%)

Time

of in

vitro

culture

(days)

Starting

autonomous

use of

dilators

(days)

Sexual

activity

initiation

(weeks)

FSFI score

(min. 2.0-

max 36)

CASE 1 28 1 80 18 90 15 16 12 24.5

CASE 2 17 0.5 85 19 90 20 15 8 24

CASE 3 17 1.5 90 20 100 18 14 6 25.7

CASE 4 24 1 95 21 70 23 14 8 26.9

CASE 5 20 1 100 22 100 19 13 6 28.1

CASE 6 25 1.5 105 23 80 15 11 4 29.3

CASE 7 49 1 110 24 90 20 12 16 23.3

CASE 8 18 0.5 110 25 80 27 13 8 30.5

CASE 9 22 2 100 23 80 22 12 10 29.3

CASE 10 18 1.5 95 22 80 21 10 10 24.5

CASE 11 18 1.5 90 20 70 23 9 10 25.7

CASE 12 29 2 85 19 70 22 9 8 28.1

CASE 13 17 1.5 80 18 90 20 11 6 30.5

CASE 14 19 2 90 18 80 19 12 6 25.7

CASE 15 20 2 105 25 80 16 11 4 23.3

CASE 16 20 1.5 110 23 100 23 10 4 29.3

CASE 17 17 1.5 80 21 70 18 13 4 30.5

CASE 18 18 2 85 24 80 28 12 4 28.1

CASE 19 23 1 95 25 70 20 11 6 25.7

CASE 20 21 1,5 100 20 80 21 10 5 29.3

CASE 21 19 0,5 80 25 100 22 12 4 28.1

CASE 22 20 2 85 20 90 18 13 6 30.5

CASE 23 22 1,5 110 20 70 19 15 6 24.5

MEAN 21.7 1.4 94 21.5 83 20.4 12 7 27.2

MANUSCRIP

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ACCEPTED

ACCEPTED MANUSCRIPT

MANUSCRIP

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ACCEPTED

ACCEPTED MANUSCRIPT

http://www.AAGL.org/jmig-22-2-JMIG-D-14-00294

Autologous in vitro cultured vaginal tissue for vaginoplasty in women with...

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