Keratinocyte growth factor and its receptor in human ovaries from fetuses, girls and women.

Keratinocyte growth factor (KGF) promotes growth of rat pre-antral follicles. There is limited information regarding its presence or that of its unique receptor (KGFR) in human ovaries, specifically in pre-antral follicles. The aim of the study was to investigate the expression of KGF and KGFR in ovarian samples from human fetuses and girls/women. The samples were prepared for immunohistochemical study of the KGF protein and for in situ hybridization to localize mRNA transcripts of KGFR. Total RNA was extracted from frozen ovarian samples, and the expression of KGF mRNA transcripts was investigated by reverse transcriptase polymerase chain reaction. In both fetuses and girls/women, the protein for KGF was detected from primordial stages in oocytes, granulosa cells (GCs) and stroma cells. Its mRNA transcripts were also detected in all extracts. The mRNA transcripts for KGFR were detected mainly in stroma cells in ovarian samples from both sources; in 10% of the samples, follicular staining was noted also in oocytes and GCs. Further studies adding KGF to the culture medium are needed to elucidate its putative role in human primordial follicle activation.

Platelet-derived growth factors (PDGF-A and -B) and their receptors in human fetal and adult ovaries.

There is no information regarding the presence of platelet-derived growth factors (PDGFs) and their receptors in human ovaries. The expression of PDGF-A, -B and their two receptors, PDGFR-alpha and -beta, was investigated in ovarian samples from women/girls and from human fetuses, at the protein and mRNA levels. The samples were prepared for immunohistochemical staining for PDGF-A and -B and their two receptors and in situ hybridization for the detection of the mRNA transcripts of the receptors. Total RNA was extracted from frozen ovarian samples, and the expression of PDGF-A and -B was investigated by reverse transcription-polymerase chain reaction. The proteins for PDGF-A and -B were detected in oocytes, and in granulosa cells (GC) of 50% of the follicles from women/girls. The proteins and mRNA transcripts for the two receptors were detected in oocytes (mRNA for PDGFR-beta only in 25% of the oocytes). PDGFR-alpha mRNA was expressed in GC of a minority of the samples from women/girls, whereas PDGFR-beta protein and mRNA were identified in over 50% of the GC from this source. PDGF-A and -B transcripts were identified in all the extracts. The presence of the receptors in GC suggests that PDGFs might be involved in the activation of primordial follicles.

In vitro maturation of human primordial ovarian follicles: clinical significance, progress in mammals, and methods for growth evaluation.

As cancer treatment improves, more young women of reproductive age are surviving, but they suffer from infertility as a consequence of the radiation and chemotherapy. Human ovarian tissue containing immature primordial follicles has been successfully cryopreserved. The ultimate aim of this technique is to induce ovarian function by re-plantation of ovarian tissue or, further into the future, by in vitro maturation (IVM) of the oocytes derived from the cryopreserved-thawed ovarian tissue, followed by routine in vitro fertilization. IVM of primordial follicles from young cancer survivors would avoid the risk of cancer re-transmission by the ovarian grafts. The present review discusses the current achievements in IVM of female germ cells and primordial ovarian follicles and the attempts to improve their development by adding various factors to the culture medium. The established methods for the evaluation of survival and growth in culture are also discussed: follicular counts, immunocytochemical methods, transmission electron microscopy, fluorescent viability markers and endocrine assays. Although the development of IVM systems is still in its infancy, researchers need to pursue their approach step-by-step, especially with regard to factors that might be involved in the activation of the ovarian follicles or female germ cells. The final measure of success will be the ability of the in vitro matured oocytes to fertilize and produce healthy offsprings. The availability of such treatment will probably lead to its demand not only by cancer patients but by other women as well.

Presence of NGF and its receptors in ovaries from human fetuses and adults.

The ability to mature human primordial follicles in vitro would assist fertility restoration. However, the signals initiating growth of primordial follicles are unknown. Growth factors such as nerve growth factor (NGF) may play a role in this process. To investigate the expression of NGF and its receptors, p75 and TrkA, in early developing follicles (mostly primordial, primary and secondary follicles), ten ovarian samples from adolescents/adults aged 13-39 and 33 ovaries from human fetuses aged 19-33 gestational weeks (GW) were obtained and immediately fixed or frozen. The fixed samples were prepared for a study of immunocytochemical staining of NGF and its two receptors. Total RNA was extracted from the frozen ovarian samples, and the expression of NGF, TrkA and p75 was investigated by RT-PCR. Products were resolved by 1% agarose gel electrophoresis and image analysis. Immunocytochemical staining revealed the expression of NGF in granulosa cells (GC) and oocytes; TrkA was mainly in oocytes and in GC in minority of the samples; and p75 was in some of the stroma cells from fetuses aged less than 22 GW. Transcripts of NGF and TrkA were identified by RT-PCR in all samples, while those for p75 were detected only in ovarian samples from fetuses aged less than 22 GW. To elucidate if NGF is indeed involved in growth initiation of human primordial follicles, it should be added to their culture medium. The immunocytochemical detection of p75 in some of the stroma cells and transcripts in ovarian samples of fetuses less than 22 GW may suggest its role in follicular assembly.

Immunocytochemical detection and RT-PCR expression of leukaemia inhibitory factor and its receptor in human fetal and adult ovaries.

The ability to mature human primordial follicles in vitro would assist fertility restoration. However, the signals initiating growth of primordial follicles are unknown. Growth factors such as leukaemia inhibitory factor (LIF) may play a role in this process. To investigate the expression of LIF and its receptor in early developing follicles, nine ovarian samples from adolescents/adults aged 13-43 years and 23 ovaries from human fetuses aged 19-33 gestational weeks were immediately fixed or frozen. The fixed samples were prepared for a study of immunocytochemical staining of LIF and its two receptor units (LIF-R and gp 130). mRNA was extracted from the frozen ovarian samples, and the expression of LIF, LIF-R and gp 130 was investigated by RT-PCR. Products were resolved by 10% polyacrylamide gel electrophoresis and image analysis. There was strong to moderate immunocytochemical staining for LIF and LIF-R in oocytes from the primordial follicular stages onwards, and very weak to moderate staining for gp 130. LIF-R was also detected in granulosa cells of primary and secondary follicles from adolescents/adults. Transcripts of LIF, LIF-R and gp 130 RNA were identified by RT-PCR in all samples. The immunocytochemical staining and mRNA expression of LIF and its receptor are consistent with the concept that LIF might be involved in growth initiation of human primordial follicles through its receptor.

Turner's syndrome and fertility: current status and possible putative prospects.

Women with Turner's syndrome should be carefully followed throughout life. Growth hormone therapy should be started at age 2-5 years. Hormone replacement therapy for the development of normal female sexual characteristics should be started at age 12-15 years and continued for the long term to prevent coronary artery disease and osteoporosis. Most women with Turner's syndrome have ovarian dysgenesis; therefore, they are usually infertile, and in very rare cases have spontaneous menses followed by early menopause. Only 2% of the women have natural pregnancies, with high rates of miscarriages, stillbirths and malformed babies. Their pregnancy rate in oocyte donation programmes is 24-47%, but even these pregnancies have a high rate of miscarriage, probably due to uterine factors. A possible future prospect is cryopreservation of ovarian tissue containing immature follicles before the onset of early menopause, but methods of replantation and in-vitro maturation still need to be developed. Should these autologous oocytes indeed be used in the future, affected women would need to undergo genetic counselling before conception, followed by prenatal assessment.

Morphological study of fully and partially isolated early human follicles.

OBJECTIVE: To compare the development of fully and partially isolated human follicles by using various culture systems. DESIGN: Human ovarian material was incubated with collagenase and deoxyribonuclease. Fully and partially isolated follicles (30-50 microm) were dissected and studied under light and electron microscopy. The follicles were then cultured on and within various matrices. Fully isolated follicles were also cocultured with stromal cells. SETTING: Rabin Medical Center, a major care and referral center. PATIENT(S): Women undergoing laparoscopy. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Microscopy studies, follicular measurements. RESULT(S): Electron microscopy studies revealed an excess of lipid droplets in the granulosa cells of freshly isolated follicles. An increase in follicular size and granulosa cell number was observed only in the fully isolated follicles cultured within collagen gels for 24 hours. Most of the partially isolated follicles detached from the collagen gels. When cultured on collagen, extracellular matrix, and poly-L-lysine, both the fully and the partially isolated follicles deteriorated within the first 24 hours; coculture with stromal cells had no beneficial effect. CONCLUSION(S): The excess in lipid droplets in granulosa cells of isolated follicles might suggest that the isolation process does not yield completely healthy follicles. However, despite this finding, our studies show that fully isolated follicles, but not partially isolated follicles, can grow within, but not on, a culture matrix.

[Microlaparoscopy in diagnostic and operative gynecologic procedures].

A recent further development in laparoscopic surgery is microlaparoscoopy. The technique is identical to standard 10 mm laparoscopy except for the use of small, 2 mm scopes and trocars. We used this technique in 19 patients treated from June 1998 to February 1999. The advantages of microlaparoscopy are: smaller incisions, less risk of damage to pelvic organs and less postoperative pain. However, the use of microlaparoscopy for operative procedures is limited to simple operations due to the limited field of vision. We urge our colleagues to consider microlaparoscopy whenever diagnostic laparoscopy is needed.

Scanning electron microscopy and X-ray diffraction studies in the analysis of medical materials: Gore-Tex versus braided polyester tape for repair of the incompetent cervix.

Cathodoluminescence in the scanning electron microscope and roentgenogram diffraction studies were applied to the analysis of two different medical materials used in cervical cerclage, the treatment of cervical incompetence. Braided polyester tape or Gore-Tex was used as suture material and was analyzed both before and after use in cerclage of the cervix. Results of SEM and X-ray diffraction studies showed distinct differences between the two materials, with the Gore-Tex demonstrating a superior morphological integrity and a decreased likelihood of phagocyte adhesion. These results correlated well with the clinical observations, which also showed Gore-Tex to be a superior medical material.

Pilot study of isolated early human follicles cultured in collagen gels for 24 hours.

The human ovarian cortex contains mainly primordial and primary follicles. The ability to mature these follicles in vitro could be of great importance for infertility treatments. Fresh and frozen-thawed ovarian tissue was incubated with collagenase and DNase. Follicles with one layer or an incomplete second layer of granulosa cells were then dissected. The follicles were embedded in collagen gels and cultured with Earle's balanced salt solution, 10% fetal calf serum and 0.5 IU/ml follicle stimulating hormone. Increases in the number of granulosa cell layers and in oocyte size were observed in 40 and 38.7% of the follicles from fresh and frozen-thawed tissue respectively, during a 24 h culture period. All the growing follicles were surrounded by cellular outgrowths. Attempts to culture the follicles longer resulted in deterioration of the follicles and oocyte release. Since our study was purely morphological, further growth parameters, e.g. DNA synthesis, should be examined in the future.

Cervical pregnancy--a conservative stepwise approach.

A case of cervical pregnancy resistant to intramuscular methotrexate therapy is presented, which was successfully treated by intra-arterial methotrexate followed by selective prophylactic hypogastric artery embolization to avoid aggravating the vaginal bleeding. It is suggested that, in cervical pregnancies in which fertility preservation is desired, a stepwise conservative approach should be applied before resorting to surgical intervention.

The usefulness of iodine-123 whole-body scans in evaluating thyroid carcinoma and metastases.

OBJECTIVE: It is recognized that diagnostic doses of (131)I larger than 3 mCi will cause some cell injury to the tissue in which it concentrates and reduce subsequent uptake of (131)I administered therapeutically. Iodine-123 has been suggested as an alternate radiopharmaceutical to perform whole-body scans since its primary emissions are photons with minimal particulate radiation and it does not cause thyroid stunning and cell injury. The purpose of this study was to assess the effectiveness of (123)I for whole-body scans. METHODS: We examined 12 patients who had (123)I whole-body scans for known papillary/follicular thyroid cancer and 1 patient with Hurthle cell carcinoma, all with suspected metastases. All patients had undergone neck surgery and were given 0.81-0 mCi (123)I. Twenty-four hours later a whole-body image and static views of relevant areas were obtained. If abnormal uptake was noted, patients were treated with large doses of (131)I and then had whole-body (131)I scans 7-10 d post-therapy. These images were compared to (123)I whole-body scans. RESULTS: All 13 patients had abnormal (123)I scans and were treated with therapeutic doses of (123)I, followed by whole-body scans 7-10 d later. In 11 patients the activity seen on the 123I scans correlated well with that seen on (131)I scans. In 1 patient, additional lesions were noted on the (131)I images. In another patient, neck activity was seen on the (123)I scan but not on the subsequent (131)I post-therapy scan. The (123)I activity was felt to represent esophageal lumen activity. CONCLUSION: We found (123)1 effective in demonstrating residual thyroid tissue, thyroid carcinoma and metastases, and recommend its use for whole-body iodine scans since it does not cause thyroid stunning.

Laparoscopic management of rudimentary horn pregnancy.

A unicornuate uterus with rudimentary horn is a rare Müllerian abnormality. This uterine anomaly may cause many gynaecological and obstetric complications, including infertility, recurrent abortions, preterm deliveries and rupture of the uterus, especially when the pregnancy implants in the rudimentary horn. To date, laparotomy has been the treatment of choice when resection of a rudimentary horn was indicated. We report on the case of a woman who benefited from laparoscopic surgery of a rudimentary horn pregnancy. Laparoscopy, in these exceptional cases, is the most accurate diagnostic tool that carries significant advantages in effective surgical management, thereby avoiding laparotomy.

Preservation of fertility in women undergoing chemotherapy: current approach and future prospects.

PURPOSE: Anticancer treatment causes ovarian failure. METHODS: Some hormones may have a protective effect on the ovary. Cryopreservation (freezing) of oocytes has had very limited success, and therefore, currently its use before chemotherapy is not a feasible option. However, cryopreservation of embryos is possible. Another solution is oocyte donation followed by in vitro fertilization (IVF). RESULTS: Ovarian cortical slices containing primordial follicles have been cryopreserved successfully. To restore fertility, cryopreserved-thawed tissue taken from cancer patients before therapy could be replanted after recovery. The possible risk of malignancy restoration could be eliminated by obtaining unilaminar follicles from cryopreserved-thawed tissue and growing them in vitro, followed by routine IVF. CONCLUSIONS: Although women who undergo chemotherapy face limited options for fertility preservation, intensive studies in cryopreservation and in vitro maturation of follicles harbor hope for brighter prospects in the future.

Abnormal sonographic endometrial findings in asymptomatic postmenopausal women: possible role of antihypertensive drugs.

OBJECTIVE: The purpose of this study was to evaluate the possible effect of the antihypertensive agent nifedipine (a calcium channel antagonist) on the endometrium of asymptomatic menopausal women by using transvaginal sonography, hysteroscopy, and endometrial sampling. DESIGN: Eighty-five asymptomatic postmenopausal women with an ultrasonographically detected endometrial thickness of more than 7 mm were examined. The study group comprised 43 women treated with nifedipine (30-60 mg/day) for mild to moderate hypertension for at least 1 year, and the control group comprised 42 normotensive women. All participants underwent transvaginal sonography, hysteroscopy, and, if necessary, endometrial biopsy. RESULTS: Of the 85 patients, 53 were found to have atrophic endometrium, 36 (83.7%) in the study group and 17 (40.5%) in the control group. The difference between the groups was statistically significant (p < 0.05). There were three cases of hyperplasia and three cases of carcinoma of the endometrium, with similar rates in the two groups. CONCLUSIONS: Among asymptomatic postmenopausal patients with sonographically thickened endometrium, atrophic endometrium was found to be significantly more prevalent in those using calcium channel antagonists to control hypertension than in those who were normotensive controls. The presence of endometrial hyperplasia and malignancy in the study group underscores the importance of meticulous endometrial evaluation whenever sonographically thickened endometrium is encountered.

Use of MRI in evaluating postoperative changes in patients with bone and soft tissue tumors.

We reviewed the clinical, surgical and magnetic resonance imaging (MRI) findings in 80 patients who underwent resection of primary benign or malignant bone or soft tissue tumors. There were 18 benign and 62 malignant tumors. Although 31 patients were originally thought to have recurrence, on review only 20 patients were considered to have recurred. Of these, three were found only to have postoperative changes at surgery. Seventeen patients actually had recurrence of tumor. We believe that the presence of an actual mass lesion on MRI is the cornerstone for the correct diagnosis of recurrent tumor.

The management of dermoid cysts--a comparative study of laparoscopy and laparotomy.

The aim of our study was to compare laparoscopy with laparotomy for the removal of ovarian dermoid cysts. Thirty-eight women with benign ovarian dermoid cyst were allocated for either laparoscopy (18 patients) or laparotomy (20 patients). The two groups were compared for operative and hospitalization times and postoperative course. Operating time was longer (93.6 +/- 23.8 min) and hospitalization time significantly shorter (22.4 +/- 6.6 h) in the laparoscopy group. No complications were reported in either group. We conclude that operative laparoscopy is a safe procedure for the removal of dermoid ovarian cysts and is as effective as laparotomy.

Conservative management of postmenopausal pelvic echo-free findings--preliminary results.

OBJECTIVES: To develop a conservative protocol for the treatment of echo-free findings in the pelvis, diagnosed by sonography in post-menopausal women, in order to avoid unnecessary surgical intervention. METHODS: A prospective study where each post-menopausal-patient underwent sonographic evaluation of the pelvic mass, serum Ca-125 levels, ambulatory aspiration of the cyst, and operative laparoscopy if necessary. RESULTS: One hundred and eighty-two women were included in the 3 years of the study. Only two cases of malignancy were diagnosed. There were no false-negative results. CONCLUSION: Although the results of our protocol of conservative management look promising, we cannot draw final conclusions because of the small sample size. Perhaps as our knowledge increases, physicians will feel more confident in handling adnexal findings conservatively.