In vitro survival of follicles in prepubertal ewe ovarian cortex cryopreserved by slow freezing or non-equilibrium vitrification.

PURPOSE: Vitrification is a well-accepted fertility preservation procedure for cryopreservation of oocytes and embryos but little is known regarding ovarian tissue, for which slow freezing is the current convention. The aim of the present study was to assess the efficiency of non-equilibrium vitrification compared to conventional slow freezing for ovarian cortex cryopreservation. METHODS: Using prepubertal sheep ovaries, the capacity of the tissue to sustain folliculogenesis following cryopreservation and in vitro culture was evaluated. Ovarian cortex fragments were cultured in wells for 9 days, immediately or after cryopreservation by conventional slow freezing or non-equilibrium vitrification in straws. During culture, follicular populations within cortex were evaluated by histology and immunohistochemistry for PCNA and TUNEL. Steroidogenic activity of the tissue was monitored by assay for progesterone and estradiol in spent media. RESULTS: No significant differences in follicle morphology, PCNA, or TUNEL labeling were observed between cryopreservation methods at the initiation of culture. Similar decreases in the proportion of primordial follicle population, and increases in the proportion of growing follicles, were observed following culture of fresh or cryopreserved ovarian tissue regardless of cryopreservation method. At the end of culture, PCNA and TUNEL-positive follicles were not statistically altered by slow freezing or vitrification in comparison to fresh cultured fragments. CONCLUSIONS: Overall, for both cryopreservation methods, the cryopreserved tissue showed equal capacity to fresh tissue for supporting basal folliculogenesis in vitro. Taken together, these data confirm that both non-equilibrium vitrification and slow-freezing methods are both efficient for the cryopreservation of sheep ovarian cortex fragments.

Uterine allotransplantation in ewes using an aortocava patch.

BACKGROUND: We investigated a novel allotransplantation model using an aortocava patch in ewes. METHODS AND RESULTS: We carried out 10 uterine orthotopic allotransplantations in ewes with end-to-side anastomosis of the aortocava donor patch on the left external iliac vessel recipient. The immunosuppressive protocol was a combination of cyclosporine (10 mg/kg/day) and mycophenolic acid (3 g/day). An estimation of the immunosuppressive therapy exposure was performed by measuring the area under the curve (AUC) of immunosuppressive plasma concentrations. The graft was assessed by vaginoscopy, magnetic resonance imaging (MRI) and second look laparotomy at 6, 8 and 10 weeks, respectively. The median (range) times for cold and warm ischemia were 95 min (75-130) and 91 min (55-165), respectively. All the vascular anastomoses were patent at the end of the surgery. The median AUC of cyclosporine and mycophenolic acid were 1.24 mg h/l (0.34-3.85) and 18.40 mg h/l (3.76-42.35), respectively. Of the 10 ewes receiving a transplant, 6 could be assessed. Cervical biopsies showed signs of necrosis in all six ewes. The MRI results correlated with the macroscopic observations of the 'second look' laparotomy. The aortocava vascular pedicles were thrombosed, adding to the peripheral neovascularization. Graft histology showed endometrial tissue in two out of six ewes. CONCLUSIONS: Mobility of the transplant within the pelvis, the length of the vascular pedicle and rejection can explain the high rate of transplant necrosis. The particular digestive anatomy and physiology of ruminants makes it difficult to administer an optimal immunosuppressive treatment. MRI appears to be a good non-invasive examination for graft estimation.

Robot-assisted laparoscopic auto-graft of patchwork ovarian cortex in two steps.

The main difficulty of ovarian tissue autograft (OTA) is to preserve as many follicles as possible because the ovarian tissue undergoes warm ischemia during grafting until revascularisation, resulting in significant follicular loss. We describe a two-stage grafting technique to stimulate new vascularisation in order to enhance the revascularization process to reduce the ischemic injuries. Furthermore we performed ovarian patchwork in the laboratory and then grafting with robotic laparoscopy to facilitate surgery and increase precision. This technique is used in the DATOR study with promising results, such as a 40% delivery rate.

[Uterine transplantation: animal and human studies]. / Transplantation de l'utérus : expériences animales et humaines.

Many cases of not life saving transplanted organs were described with the aim of improving quality of life. Uterus graft could be an alternative solution to adoption or surrogacy for women who have uterine factor infertility. Different animals' studies with mouse, sheep or monkey showed feasibility of the surgical technique with large vessels patch. One case of human uterine transplant has been reported but failed. Cold storage of the uterus in protective solution has been explored with mouse, sheep and human. Only pregnancy after uterus graft by syngenic mouse has been published. Results about pregnancy after allograft with sheep or monkey are necessary before pregnancy after human uterus graft becomes a reality.

[Imaging of female infertility]. / Imagerie de l'infertilité féminine.

Medical treatment of a couple's infertility depends upon the cause(s) of infertility. The goals of imaging are to determine these causes (male, female, mixed, unexplained), to evaluate their severity and type on the female side central, ovarian, uterine, tubal, peritoneal, endometriosis, in order to propose an adapted treatment. Imaging modalities are essentially non ionising (US and MRI). Hysterosalpingogram retains its indication to study tubal patency, it can be completed by tubal catheterisation in cases of proximal tubal obstruction.

Uterus transplantation in France: for which patients?

OBJECTIVE: Uterine infertility (UI), which can be caused by a variety of congenital or acquired factors, affects several thousand women in Europe. Uterus transplantation (UTx), at the current stage of research, offers hope for these women to be both the biological mother and the carrier of their child. However, the indications of UTx still need to be defined. The main aim of the study was to describe the different etiologies of UI and other data as marital and parental status from women requesting UTx who contacted us in the framework of a UTx clinical trial. Secondarily, we discussed the potential indications of UTx and their feasibility. STUDY DESIGN: This is an observational study. RESULTS: Of a total of 139 patients with UI, 105 patients (75.5%) had uterine agenesis, making it the leading cause of UI in this sample. Among the patients with uterine agenesis, 25% had a solitary kidney and 44.7% had undergone vaginal reconstruction. Peripartum hysterectomy, hysterectomy for cancer, and hysterectomy for benign pathologies accounted for 9.4%, 7.2% and 5% of cases, respectively. Less common causes of UI included complete androgen insensitivity syndrome (2.2% of patients) and prenatal diethylstilbestrol exposure (0.7%). Approximately 14% of the women already had at least one child and 66% were in a couple living together for at least 2 years. CONCLUSION: UTx is still under evaluation and further research is under way. Nulliparous patients with no major medical or surgical history and with normal ovarian function, who meet the legal criteria for medically assisted reproduction, represent the best indications for UTx at this stage of its development.

[Uterine factors limiting ART coverage]. / Facteurs utérins limitant la prise en charge en AMP.

Transvaginal sonographic evaluation of endometrial texture and thickness may be an indicator of the likelihood of achieving pregnancy. Very few pregnancies occur when the endometrial thickness was less than 6 mm. When type C endometrium (multilayered endometrium consisting of prominent outer and midline hyperechogenic lines and inner hypoechogenic regions) greater than or equal to 6 mm thick was seen, the pregnancy rate per embryo transfer is goods. MR imaging is highly accurate in helping to diagnose adenomyosis which is a cause of repeated implantation failures in women undergoing IVF. Furthermore the MRI evaluation of junctional zone thickness (AJZ>7 mm and MJZ>10 mm) is the best negative predictive factor of implantation failure (97%). Thus, we advocate the measurement of junctional zone thickness with MRI in women with 2 consecutive implantation failure following IVF.

[Uterine transplantation: is there a real demand?]. / Transplantation utérine : une demande réelle ?

OBJECTIVES: To study the demand there is for uterus transplantation (UTx). PATIENTS AND METHODS: Recent media coverage of developments in UTx prompted associations of patients with Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome and of women suffering from UI to contact us. We sent them anonymous questionnaires devised to sound out their attitude towards UTx and towards adoption and gestational surrogacy (GS). A clinical psychologist also carried out a qualitative discourse analysis. RESULTS: Sixty patients answered the questionnaire. Thirty-eight patients were married or living with a male partner. Seven patients had had a hysterectomy. Fifty-one patients had uterine agenesis. Of the 60 patients, 19 and 21, respectively, had ruled out the option of adoption or GS, and 11 would not envisage either possibility. Thirty-five patients were willing to take part in a clinical study into UTx despite the uncertainty of the outcome and the potential risks involved. Of these 35 volunteers, 23 were in a heterosexual relationship and aged ≤35 years. DISCUSSION AND CONCLUSION: For women with UI the condition is all the more distressing because there is no medical solution for it. UTx could hold out hope for some of these patients despite the complexity of the procedure and the attendant risks. Because of the feelings of vulnerability engendered by UI, any UTx programme should provide full information to patients and ensure they are carefully screened and selected.

Laparoscopically assisted vaginal hysterectomy for non-malignant disease of the uterus. Report on a personal series of 126 cases.

OBJECTIVE: A report is given of a series of 126 laparoscopically-assisted vaginal hysterectomies (LAVH) for benign lesions, carried out between September 1990 and December 1995. MATERIALS AND METHOD: The mean age of the patients was 50.3 years, and the main indications for hysterectomy were metrorrhagia (88). The main reasons why LAVH was chosen from among other hysterectomy techniques were a large uterus (55), associated ovarian surgery (45), and a difficult vaginal approach (35). The surgical technique always began with a laparoscopic stage followed by a vaginal stage. The laparoscopic stage generally finished at the lower part of the broad ligament. The vagina was opened and the uterine arteries were ligatured by a vaginal approach (116). Only 10 total laparoscopic hysterectomies were performed. RESULTS: The mean duration of the operation was 72 +/- 28 min, mean blood loss was 1.89 g/dl, and mean uterus weight was 224 g (maximum = 1093 g). Operative complications consisted of two bladder wounds and two switches to abdominal hysterectomy. Postoperative complications were urinary infections (17), hemorrhages needing second-look operations [2] and abscess of the vaginal section requiring evacuation [3]. CONCLUSION: LAVH should never be carried out instead of vaginal hysterectomy (VH), since VH is the best procedure when it is easy to perform. The authors use LAVH when VH is difficult or contraindicated (the aim being to avoid laparotomy) and actually carry out less than 5% of hysterectomies for benign lesions by laparotomy.

Ovarian tissue cryopreservation and gynecologic oncology: a review.

Sperm cryopreservation permits young men, undergoing cancer treatments, to preserve their fertility. Ovarian tissue cryopreservation have the same goal for young women and could also be an option for children. However, only primordial follicles survive after freezing and a follicular maturation is needed after thawing. This maturation has not yet been realized in humans, pregnancies have only been obtained in animal models. As cryopreservation is yet effective in humans, many teams have already cryopreserved the ovarian tissue of patients who have nothing to lose as their follicular reserve would have been destroyed or severely depleted by cancer treatment. The preservation of fertility is rarely an issue in gynecologic oncology because it usually concerns post-menopausal women. However, they are early-onset forms of gynecologic cancers and in these cases fertility is often threatened. Ovarian tissue cryopreservation may be performed when curative or prophylactic ovariectomy must be undergone, when chemotherapy with high-dose alkylating agents is planned or when pelvic radiation is needed (particularly in cases requiring chemotherapy combined with radiotherapy). In some of these situations it would be dangerous to graft back the tissue to the patient as cancer cells could remain within the grafts, the best solution in this case would be the in vitro follicular maturation.

[Cryopreservation of ovarian tissue]. / La congélation du tissu ovarien.

Ovarian tissue cryopreservation (OTCP) is a new procedure of medically assisted procreation, still at the experimental stage, whose primary aim is to store female gametes as sperm cryopreservation permits to do for male gametes. Ovarian tissue is removed very simply by laparoscopy. It survives well to freezing if the medium contains a cryoprotective agent and the rate of freezing is slow. In contrast, thawing must be rapid. There are three processes for the utilization of ovarian tissue after thawing. In vitro maturation and xenografting remain impossible for technical and ethical reasons. Autologous transplantation (orthotopic or heterotopic) of the tissue is therefore the only foreseeable method over the short term. Indications for OTCP must remain rare as long as no pregnancy has been obtained in human. At the present time, only female patients who would inevitably suffer the loss of their fertility should be able to take advantage of OTCP. Basically, this would mean women subjected to castrating anticancer therapy. It would seem reasonable to set the age limit at 35-years for carrying out OTCP. Lastly, female patients should be clearly informed that the method is still at the research stage, and in France samples must be taken in accordance with the laws governing clinical research.

[Uterine Doppler study for pre-implantation prognosis of the embryo]. / Doppler utérin dans le pronostic pré-implantatoire de l'embryon.

To assess if a simple US criteria was present allowing pre- and post-implantation evaluation of the quality of the embryo. Measurement of the pulsatility index (PI) of the uterine arteries in association with evaluation of the type of curves was useful for follow-up. PI correlates with the number of follicules, the number of cells at Day 2, and the likelihood of pregnancy whereas the type of curve directly correlates with the appearance of the oocytes and embryo before the transfer. These results should be confirmed by larger studies but they could lead to early detection and a treatment of these abnormalities of vascularization.

[Heterotopic pregnancy: a report of 5 cases and review of the literature]. / Grossesse hétérotopique: à propos de 5 cas et revue de la littérature.

Heterotopic pregnancy, also called combined pregnancy, associates extra-uterine pregnancy and intra-uterine pregnancy. We report five cases of heterotopic pregnancy illustrating this condition and reviewed the recent literature from 1994 to 1999. We compared the results of this review with earlier data in the literature from 1971 to 1993. We observed that therapeutic management of the extra-uterine pregnancy and course of the intra-uterine pregnancy have changed little over this period. Conversely, the frequency of heterotopic pregnancy has steadily increased since the development of medically assisted reproduction, although spontaneous cases continue to occur. The diagnosis of heterotopic pregnancy is often difficult as the symptomatology is often misleading. Transvaginal ultrasound generally gives the diagnosis which may be confirmed by laparoscopy, allowing treatment of the extra-uterine pregnancy. Maternal and intra-uterine fetal prognosis depends on early diagnosis which should be made, if possible, prior to termination of the extra-uterine pregnancy.

[Folates and the neural tube. Review of the literature]. / Folates et tube neural. Revue de la littérature.

There are two types of neural tube fusion abnormality (NTFA)--craniorachischisis and rachischisis--with a prevalence of 11.2/10,000 in continental Europe. Their prevalence varies widely and is influenced by many factors, including geographical, racial and seasonal components. The transmission of NTFAs is usually polygenic and affected by many factors, and far more rarely of the mendelian monogenic type. The occurrence of NTFAs has been shown to be due to folic acid (FA) deficiency. First suspected in 1965, FA deficiency was proved to be a cause of NTFAs both occurring and recurring in the 1980s. FA deficiency might act by preventing the remethylation of homocysteine to methionine, the latter is known to play an essential role in fusion of the neural tube in animals. Primary prevention of NTFAs therefore requires that FA supplementation be given to women planning a pregnancy and after conception. Two possible ways of preventing FA deficiency can be envisaged, ie. via folic acid supplementation or systemic enrichment of the basic diet. Neither of the two alternatives is sufficient by itself and probably a combination of the two would provide the best means of preventing neural tube defects.

[Indications for cryopreservation of ovarian tissue]. / Indications de la cryopréservation du tissu ovarien.

BACKGROUND: The first attempts at ovarian tissue cryopreservation (OTCP) were performed in the 1950s. Recent research efforts have demonstrated the possibility of obtaining pregnancy with this technique in three animal species and have shown good primordial human follicle survival up through the freezing process. POTENTIAL INDICATIONS: OTCP is a procedure designed to protect ovarian tissue from threats to its follicular reserves. The first threat is the time-related massive physiological destruction of the follicular reserve ending with menopause. OTCP would enable this wastage to be arrested, thereby prolonging ovarian cycling beyond limits. Conditions producing premature menopause, when known in advance, may also potentially benefit from OTCP. The iatrogenic destruction of the follicular reserve by radiation therapy or alkylating agents is another situation where OTCP would enable the patient's fertility to be preserved. Among these clinical settings, iatrogenic destruction of follicular stocks appears to us, with the current state of research, to be an acceptable indication for OTCP.

[Follicle freezing and autografting. A new method of medically assisted procreation?]. / Congélation folliculaire et autogreffe. Une nouvelle méthode de procréation médicalement assistée?

Although frozen human follicles were shown to be viable after thawing, more than forty years, the low yield of primordial follicles after cryopreservation has greatly limited research in this area. The aim of freezing follicles is to stock a patient's ovocytes before potentially destructive therapy for cancer then to reimplant the follicular tissue after successful treatment. The patient's ovulatory capacity would then theoretically be restored, allowing natural or possibly in vitro fertilization. As have other teams, we recently renewed work in cryopreservation of follicles in experimental models in search for a better cryopreservation agent and have had encouraging results suggesting that primordial human follicles or ovarian fragments can be grafted successfully after cryopreservation. Questions still under study include the optimal site for implantation, the survival time for grafts and the quantity of follicles needed to achieve pregnancy. Work on large animal models or xenografts of human tissues on immunotolerant animals may provide further insight. The possibilities of grafting primordial follicles, either alone or within ovarian tissue, may raise hope for many women, although new questions such as an age limit for reimplantation and use of allografts requiring preparation with major immunosuppressive therapy require further debate.

[Ambulatory anesthesia in gynecologic surgery]. / Anesthésie ambulatoire en chirurgie gynécologique.

Surgical gynecologic procedures are among the best clinical indications of ambulatory surgery as the patients are usually young and healthy. The beneficial effects of this type of organization (economical, medical and psychological) are numerous. A rational approach of the main objectives of ambulatory surgery and anesthesia is needed to choose the more logical anesthetic agents to allow rapid patient's recovery with a minimal rate of postoperative problems. These issues are therefore briefly developed in this text.