ABSTRACT
Melanoma is one of the most common cancers in adolescents and adults at fertile age, especially in women. With novel and more effective systemic therapies that began to profoundly change the dismal outcome of melanoma by prolonging overall survival, the wish for fertility preservation or even parenthood has to be considered for a growing portion of melanoma patients-from the patients' as well as from the physicians' perspective. The dual blockade of the mitogen-activated protein kinase pathway by B-Raf proto-oncogene serine/threonine kinase and mitogen-activated protein kinase inhibitors and the immune checkpoint inhibition by anti-programmed cell death protein 1 and anti-cytotoxic T-lymphocyte-associated protein-4 monoclonal antibodies constitute the current standard systemic approaches to combat locally advanced or metastatic melanoma. Here, the preclinical data and clinical evidence of these systemic therapies are reviewed in terms of their potential gonadotoxicity, teratogenicity, embryotoxicity and fetotoxicity. Recommendations for routine fertility and contraception counseling of melanoma patients at fertile age are provided in line with interdisciplinary recommendations for the diagnostic work-up of these patients and for fertility-protective measures. Differentiated recommendations for the systemic therapy in both the adjuvant and the advanced, metastatic treatment situation are given. In addition, the challenges of pregnancy during systemic melanoma therapy are discussed.
Subject(s)
Fertility Preservation , Melanoma , Adolescent , Antibodies, Monoclonal , Female , Humans , Immunotherapy/adverse effects , Melanoma/drug therapy , Pregnancy , Proto-Oncogene Mas , Proto-Oncogene Proteins B-rafABSTRACT
PURPOSE: In addition to guidelines focusing on scientific evidence, practical recommendations on fertility preservation are also needed. METHODS: A selective literature search was performed based on the clinical and scientific experience of the authors. This article (Part II) focuses on fertility preservation techniques. Part I, also published in this journal, provides information on disease prognosis, disease-specific therapy, and risks for loss of fertility. RESULTS: Ovarian stimulation including double stimulation and freezing of oocytes is the best-established therapy providing live birth chances in women < 35 years with high ovarian reserve of around 30-40%. Ovarian tissue freezing is especially useful in young women with good ovarian, if spontaneous conception is favoured and if < 1 week until chemotherapy is provided. Data on success rates are still limited, but this further evolving technique will possibly reach similar success rates as ovarian stimulation. GnRH agonists seem to reduce the risk of premature ovarian failure up to 50%; however, the effect is possibly not long-lasting. Ovarian transposition can easily be combined with freezing of ovarian tissue and is the preferred technique before pelvic radiotherapy. Other techniques, such as in vitro maturation, are limited to women with high ovarian reserve and remain less effective. In addition, procedures such as in vitro growth of follicles, etc. are still experimental. CONCLUSIONS: Fertility preservation in women provides realistic chances of becoming pregnant. The choice of technique needs to be based on the time required, the woman's age, its risks and efficacy, and the individual preference of the patient.
Subject(s)
Fertility Preservation/methods , Ovulation Induction/methods , Adult , Female , Humans , Prospective Studies , Young AdultABSTRACT
Purpose: Official guideline coordinated and published by the German Society of Gynecology and Obstetrics (DGGG). Aim of the guideline was to standardize the diagnosis and treatment of patients with recurrent miscarriage (RM). Recommendations were proposed, based on the current national and international literature and the experience of the involved physicians. Consistent definitions, objective assessments and standardized therapy were applied. Methods: Members of the different involved societies developed a consensus in an informal process based on the current literature. The consensus was subsequently approved by the heads of the scientific societies. Recommendations: Recommendations for the diagnosis and treatment of patients with RM were compiled which took the importance of established risk factors such as chromosomal, anatomical, endocrine, hemostatic, psychological, infectious and immunological disorders into consideration.
ABSTRACT
In women, anti-Müllerian hormone (AMH) is exclusively expressed in granulosa cells and an established marker of ovarian reserve. In menstrual cycle disorders, low AMH is usually interpreted as an indicator of primary ovarian insufficiency. This study is a case series of 11 patients with hypopituitarism. AMH concentrations were on or below the age-specific 25th percentile in three of the four patients diagnosed in infancy, but not in the remaining seven patients, who were diagnosed during adolescence or later. In patients with hypopituitarism, the detection of low AMH serum concentrations can present a diagnostic pitfall and its value in the interpretation of ovarian reserve in these patients is challenging.
Subject(s)
Anti-Mullerian Hormone/blood , Hypopituitarism/blood , Hypopituitarism/diagnosis , Ovary/physiology , Adolescent , Age Factors , Biomarkers/blood , Child , Female , Humans , Infant , Ovary/diagnostic imaging , Ultrasonography , Uterus/diagnostic imagingABSTRACT
Among the family of herpes viruses, only cytomegalovirus (CMV) and, to a lesser extent, human herpes virus 8 (HHV-8) are of relevance in transfusion medicine. Due to neutropism, herpes simplex viruses (HSV) types 1 and 2 are considered to be of minor relevance. However, several reports gave evidence that a HSV DNAemia might occur and HSV could therefore be transmissible by blood products. The aim of our study was to collect data about prevalence of HSV antibodies among blood donors and to clarify whether HSV DNAemia is possible. HSV antibody states of 653 blood donors were investigated. Blood specimens of 46 patients with primary and recurrent HSV infection were tested for HSV-1 and HSV-2 DNA using TaqMan polymerase chain reaction. In 505 of the 653 blood donors HSV antibodies were detectable, most of which were HSV-1 antibodies. HSV DNA was detected in plasma, but not in peripheral blood mononuclear cells (PBMCs) of seven rather seriously ill patients with primary herpes genitalis. No HSV viraemia was detectable in otherwise healthy patients with recurrent herpes labialis. Thus, HSV DNAemia is possible, but seems to be limited to primary infections and could not be detected in the recurrent infection. Therefore, blood donors with primary herpes infection should be deferred from donation. Blood donors with recurrent HSV infection are probably not at risk of transmitting HSV, but further studies are necessary to prove this hypothesis. Detection of HSV DNA in PBMCs as described formerly could not be confirmed by this study.
Subject(s)
Blood Donors , Blood Transfusion/standards , DNA, Viral/blood , Donor Selection/standards , Herpes Simplex/virology , Herpesvirus 1, Human/isolation & purification , Herpesvirus 2, Human/isolation & purification , Plasma/virology , Viremia/virology , Adolescent , Adult , Aged , Antibodies, Viral/blood , Female , Germany/epidemiology , Herpes Genitalis/blood , Herpes Genitalis/epidemiology , Herpes Genitalis/virology , Herpes Labialis/blood , Herpes Labialis/epidemiology , Herpes Labialis/virology , Herpes Simplex/blood , Herpes Simplex/epidemiology , Herpes Simplex/prevention & control , Herpes Simplex/transmission , Herpesvirus 1, Human/genetics , Herpesvirus 1, Human/immunology , Herpesvirus 2, Human/genetics , Herpesvirus 2, Human/immunology , Humans , Leukocytes/virology , Male , Middle Aged , Polymerase Chain Reaction , Prevalence , Recurrence , Sensitivity and Specificity , Seroepidemiologic Studies , Transfusion Reaction , Viremia/epidemiology , Young AdultABSTRACT
OBJECTIVES: The endometrium carcinoma is the most frequent malignancy of the female genital tract. Approximately, 10-20% of all patients with an endometrial carcinoma are free of symptoms until the time of diagnosis. The frequent occurrence of an endometrial carcinoma in connection with intrauterine cavity fluid collection (sero- or mucometra) has been discussed controversially in literature. What are the hysteroscopic and histological findings in patients with sonographically determined endometrial fluid in postmenopause, and how should these findings be interpreted? METHODS: 74 patients, in whom endometrial fluid without bleeding disorders had been diagnosed during routine transvaginal sonography, underwent hysteroscopy conducted with a 4.5 mm optics and dilatation and curettage (D&C). The median age of the patients was 68 years with a range of 32 years. RESULTS: The simple thickness of the endometrium, i.e. single layer measured sonographically at the point of maximal thickness, was on average 5.7 mm +/- 3.6 mm (2-15 mm). Hysteroscopically, an endometrium polyp was found in 23 cases (31.1%), endometrium hyperplasia in 12 (16.2%), and an atrophy in 35 cases (47.3%). In four cases (5.4%), an endometrial carcinoma was suspected. The histological results were consistent with the hysteroscopic findings. In all instances, in which the simple endometrial thickness amounted to 3 mm or less, an atrophic endometrium was found. The frequency of intrauterine pathologies increased significantly with a greater thickness of the endometrium. In 80% of patients, a cervical stenosis existed. CONCLUSIONS: Endometrial fluid by itself, without assessment of the endometrium, does not indicate the requirement for additional histological clarification. As diagnostics, the authors suggest especially the endometrial morphology.
Subject(s)
Body Fluids , Dilatation and Curettage , Endometrium/pathology , Hysteroscopy , Postmenopause , Aged , Aged, 80 and over , Atrophy/diagnosis , Carcinoma/diagnosis , Cervix Uteri/pathology , Constriction, Pathologic/diagnosis , Endometrial Hyperplasia/diagnosis , Endometrial Neoplasms/diagnosis , Female , Humans , Middle Aged , Polyps/diagnosis , Sensitivity and SpecificityABSTRACT
This paper examines and compares necrosis in human ovarian tissue after conventional slow freezing or vitrification and ensuing xenotranplantation. Slow cryoconserved or vitrified ovarian tissue samples and fresh controls from nine patients were subcutaneously transplanted into SCID mice. The tissue samples were explanted after 6 weeks and the necrotic areas were examined by staining with Lucifer yellow SV. The size of the necrotic areas in parallel cultivated ovarian tissue samples was compared, as was necrosis in cultivated prostate tumour spheroids where the emergence of necrosis and its pathophysiological correlation have been described. Examinations showed no significant rise in the proportion of necrotic areas after slow cryoconservation/transplantation and in the controls (transplanted fresh tissue, not transplanted fresh tissue, long-term culture). The proportion of necrotic areas in the tumour spheroids was significantly higher than in the ovarian tissue. Vitrification could, after these results, be presented as an alternative to conventional slow cryoconservation.
Subject(s)
Cryopreservation/methods , Necrosis , Organ Transplantation/methods , Ovary/pathology , Adult , Animals , Cell Line, Tumor , Female , Fluorescent Dyes/pharmacology , Freezing , Humans , Isoquinolines/pharmacology , Male , Mice , Mice, SCID , Organic Chemicals , Prostatic Neoplasms/pathology , Time Factors , Transplantation, HeterologousABSTRACT
BACKGROUND: In contrast to the technique of conventional freezing, the vitrification of spermatozoa requires high cooling rates (720 000 degrees K/min), which could be damaging for spermatozoa. The aim of our study was to compare slowly frozen and vitrified spermatozoa in terms of their post-thaw DNA integrity and motility. METHODS: Semen samples were prepared according to the routine swim-up technique and divided into aliquots for comparison of fresh, conventionally frozen and vitrified spermatozoa from the same ejaculate in the presence or absence of cryoprotectants. Spermatozoa motility and DNA integrity were determined. RESULTS: The motility of spermatozoa conventionally (slowly) frozen with a cryoprotectant was similar to that recorded for spermatozoa vitrified in the absence of cryoprotectant (47 versus 52%). The DNA integrity was unaffected by the cryopreservation method or presence of cryoprotectants. CONCLUSION: The vitrification of human spermatozoa in the absence of conventional cryoprotectants is indeed feasible. The DNA integrity of vitrified sperm is comparable with that shown by standard slow-frozen/thawed spermatozoa, yet the method is quick and simple and does not require special cryobiological equipment.
Subject(s)
Cryopreservation/methods , Cryoprotective Agents , DNA Damage , Semen Preservation , Sperm Motility , Spermatozoa/physiology , Cryopreservation/standards , Feasibility Studies , Humans , Male , Time FactorsABSTRACT
AIM: The purpose of this study was to investigate the effect of soy supplementation with isoflavones on plasma hormone levels in postmenopausal women. METHODS: 16 postmenopausal women (mean age 56.21 +/- 5.01 years) were assigned to 24 weeks of dietary soy supplementation. A defined soy protein amount per day (20 g) with a low dosage of isoflavones (20 mg) was used. Plasma samples were analyzed for estradiol, FSH, LH, prolactin, testosterone and DHEAS. RESULTS: After 24 weeks of soy supplementation, plasma levels of estradiol did not increase. Gonadotropins, prolactin and the measured plasma androgens remained unchanged. We did not see any significant treatment effects. CONCLUSIONS: In the postmenopausal hypoestrogenic situation, soy protein consumption with low isoflavones does not influence endogenous hormone levels of estradiol and gonadotropins.
Subject(s)
Estradiol/blood , Gonadotropins/blood , Postmenopause , Soybean Proteins/administration & dosage , Dehydroepiandrosterone Sulfate/blood , Dietary Supplements , Female , Follicle Stimulating Hormone/blood , Humans , Isoflavones/administration & dosage , Luteinizing Hormone/blood , Middle Aged , Prolactin/blood , Testosterone/bloodABSTRACT
The aim of our investigation was to compare the ultrastructure of lipid droplets in porcine pronuclear oocytes obtained after in vitro fertilization (IVF) or parthenogenetic activation (PGA) of Metaphase II (MII) oocytes. The ultrastructure of lipids in the IVF or PGA pronuclear oocytes after vitrification was also studied. There are two kinds of lipid droplets in porcine GV oocytes: 'dark' and homogenous vesicles next to 'grey' vesicles with electron-lucent streaks. After IVF or PGA and following 16-h culture, only the presence of 'grey' vesicles was detected in IVF and PGA oocytes. Ultra-structure of lipid vesicles of PGA and IVF oocytes was similar. The PGA oocytes can be as model of the two pronuclear stage IVF oocytes.
Subject(s)
Fertilization in Vitro/veterinary , Lipid Metabolism , Oocytes/ultrastructure , Parthenogenesis , Animals , Cryopreservation/veterinary , Female , SwineABSTRACT
AIM: To establish a prospective direction for further development of the protocol for cryopreservation of ovarian tissue by direct plunging into liquid nitrogen. MATERIALS AND METHODS: Human ovarian biopsies from 20 patients (cut in approximately 0.5mm(3) pieces) were exposed to: 40% ethylene glycol+0.35 M sucrose+5% egg yolk; 40% ethylene glycol+18% Ficoll-70+0.35 M sucrose; 20% ethylene glycol+20% dimethyl sulphoxide. Cryopreservation of pieces was accomplished by plunging 0.25 ml straws or copper grids into liquid nitrogen or 0.25 ml straws into precooled (-196 degrees C) metallic powder. Thawed pieces were transferred to sucrose solution for incremental dilution of cryoprotectants. Histological observation of the tissue was performed after cryopreservation and in vitro culture was done to study hormone production ability after cryopreservation. RESULTS: Only ultrarapid cooling in ethylene glycol-sucrose-egg yolk solution protected both follicles and stroma from damage. CONCLUSION: The following parameters were established as required for a protocol of human ovarian tissue cryopreservation by direct plunging into liquid nitrogen: the vitrification medium should include ethylene glycol, disaccharide and egg yolk; ultrarapid cooling/thawing should take place using standard 0.25 straws or copper grids.
Subject(s)
Cryopreservation/methods , Nitrogen , Ovary/physiology , Adolescent , Adult , Cryoprotective Agents , Culture Techniques , Dimethyl Sulfoxide , Egg Yolk , Estradiol/biosynthesis , Ethylene Glycol , Female , Humans , Ovary/diagnostic imaging , Progesterone/biosynthesis , Solutions , Sucrose , UltrasonographyABSTRACT
Given that it has been possible to successfully cryopreserve human ovarian tissue by direct plunging into liquid nitrogen, this study was designed to establish the future direction to be taken in this line of research. Bovine oviductal epithelial fragments (as a tissue model) and large biopsy fragments (approximately 2.0 cubic mm) of human ovarian tissue were used for cryopreservation. Two protocols were tested: with permeable cryoprotectants (dimethyl sulphoxide, propylene glycol, glycerol) + egg yolk + sucrose or trehalose + a synthetic blocker of ice nucleation, Supercool X-1000; and using only permeable cryoprotectants (glycerol and ethylene glycol) + egg yolk + Supercool X-1000. The cryopreserved tissue specimens were subsequently thawed and the cryoprotectants removed by dilution in graded sucrose solutions. Both the dynamic growth and hormonal activity of the ovarian tissue pieces, vitrified using only permeable cryoprotectants, were greater than after vitrification in a mixture of permeable cryoprotectants and sucrose. Unlike the case for other reproductive tissue (spermatozoa, oocytes, embryos), these findings suggest that the cryopreservation of ovarian tissue by direct plunging into liquid nitrogen must be achieved by vitrification using only permeable cryoprotectants and agents that prevention ice formation.
Subject(s)
Cryopreservation/methods , Cryoprotective Agents/pharmacology , Disaccharides/pharmacology , Ovary/drug effects , Tissue Preservation/methods , Adult , Animals , Cattle , Clinical Protocols , Culture Techniques/methods , Estradiol/metabolism , Female , Humans , Models, Animal , Nitrogen , Ovary/anatomy & histology , Ovary/metabolism , SolutionsABSTRACT
The aim of our study was to investigate the effects of vitrification (cooling rate approximately 10000(C/min) without cryoprotectants on swim-up prepared human spermatozoa in comparison to standard conventional freezing with cryoprotectants. Motility, morphology, rate of viability and acrosome reaction of spermatozoa were evaluated. The described method of cryopreservation of human spermatozoa by direct plunging into liquid nitrogen slush without cryoprotectants was effective and could be recommended for routine IVF.
Subject(s)
Cryopreservation/methods , Semen Preservation/methods , Cell Survival , Cryoprotective Agents , Female , Fertilization in Vitro , Humans , Male , Nitrogen , Pregnancy , Sperm Motility , Spermatozoa/cytologyABSTRACT
In this paper, the occurrence of an external genital abnormality in female marmoset monkeys (fused labia) is discussed. This malformation was detected, for the first time, in a group of animals at the German Primate Center (GPC), Goettingen. The malformed vulva was completely sealed except for an opening of 1.5-2.5 mm around the urethra sufficient for urination. Because of this defect the animals were not able to copulate. As a consequence, the affected females were functionally infertile although they had a normal genital tract and a regular cycle. This vulvar abnormality was found in 12 females, offspring of 10 pairs in which either one or both came to the German Primate Center from two genetically related colonies in Munich, Germany, and one colony in Basel, Switzerland. The abnormality appeared to be recessive and inheritable from either parent. In pairs in which both animals were from one of the mentioned colonies, 45% of the female offspring were affected. In pairs where only one partner came from these colonies, 26% of female offspring had the malformation. These results indicate that avoidance of inbreeding, which is frequently performed in primate colonies, may reduce, but not eliminate the expression of abnormalities of genetic origin. Therefore selective breeding is required, and, in colonies where these recessive mutations are widespread, the development of genetic screening tests would be advantageous.
Subject(s)
Callithrix/abnormalities , Monkey Diseases/congenital , Vulva/abnormalities , Animals , Callithrix/genetics , Female , MaleABSTRACT
OBJECTIVES: The aim of our study was to investigate the role of the intact endometrium and ovaries for serum levels of insulin-like growth factor binding protein-1 (IGFBP-1) and glycodelin. STUDY DESIGN: In 35 premenopausal patients with a planned hysterectomy, serum measurements of IGFBP-1 and glycodelin were done before surgery and 1, 3, 5, and 10 days after surgery. Patients were divided into three groups according to the kind and time of operation: (1) hysterectomy with bilateral adnexectomy in the luteal phase and (2) hysterectomy without adnexectomy in the follicular phase or (3) the luteal phase. RESULTS: IGFBP-1-we could not show any differences in IGFBP-1 serum levels before and after hysterectomy with or without bilateral oophorectomy. Glycodelin-hysterectomized and oophorectomized patients showed decreasing serum levels up to day 3. After day 5, circulating concentrations of glycodelin increased continuously but remained below pre-operative levels. In both non-adnexectomized groups we saw a reduction up to day 5 but a rise at day 10. None of the results reached statistical significance. CONCLUSION: The results indicate that endometrium and ovary are not the only sources of IGFBP-1 and glycodelin.
Subject(s)
Glycoproteins/blood , Hysterectomy , Insulin-Like Growth Factor Binding Protein 1/blood , Ovariectomy , Pregnancy Proteins/blood , Premenopause , Adult , Female , Follicular Phase , Glycodelin , Humans , Luteal Phase , Middle AgedABSTRACT
Transvaginal hydrolaparoscopy (THL) was evaluated in comparison with the already established chromolaparoscopy in the detection of tubal factors, adhesions as well as endometriosis. 43 infertile patients without previous pelvic operations and with an inconspicuous clinical examination were included in a prospective comparative study of THL and chromolaparoscopy. THL succeeded in 40 patients (93.0%). Both methods showed 100% agreement with regard to tubal factors and adhesions. However, only 72/80 tubes (90.0%) could be portrayed by THL. In contrast to this, THL failed to identify 8 of 10 laparoscopically verified endometrioses (isolated endometriosis of the bladder peritoneum in 2). No complications occurred with THL. THL could be the method of choice for the clarification of mechanical infertility factors in symptom-free patients with no suspicion of pelvic pathologies. Tubal pathologies and/or adhesions (visible during THL) should be indications for laparoscopy. In the case of inconspicuous genitals during THL and a still unfulfilled desire for offspring postoperatively, laparoscopy should be considered in order to exclude the possibility of unidentified endometriosis. Retroflexio uteri should at least be a relative contraindication for THL. Further studies are necessary to evaluate the role of THL in the diagnostic concept of infertility in the future.
Subject(s)
Infertility, Female/diagnosis , Laparoscopy/methods , Adult , Endometriosis/diagnosis , Fallopian Tube Patency Tests/methods , Female , Humans , Infertility, Female/pathology , Prospective Studies , Tissue Adhesions/diagnosisSubject(s)
Adnexal Diseases/surgery , Laparoscopy/methods , Pregnancy Complications/surgery , Pregnancy Outcome , Abdomen, Acute/diagnostic imaging , Abdomen, Acute/surgery , Adnexal Diseases/diagnostic imaging , Adult , Cesarean Section , Female , Humans , Minimally Invasive Surgical Procedures/methods , Pneumoperitoneum, Artificial , Pregnancy , Pregnancy Complications/diagnostic imaging , Pregnancy Trimester, First , Pregnancy, Multiple , Torsion Abnormality/diagnostic imaging , Torsion Abnormality/surgery , Treatment Outcome , Twins , Ultrasonography, PrenatalABSTRACT
Ectopic pregnancies sited in dehiscent cesarean section scars have a high risk of rupture and bleeding. Attempts at operative therapy frequently end in loss of the uterus. A connection with the cavum uteri justifies an attempt at dilatation and curettage. We describes a patient with combined systemic and local intra-amniotic methotrexate (MTX). The uterus was preserved.
Subject(s)
Abortifacient Agents, Nonsteroidal/therapeutic use , Cesarean Section , Methotrexate/therapeutic use , Pregnancy, Ectopic/drug therapy , Pregnancy, Ectopic/surgery , Adult , Female , Fetal Death , Humans , Pregnancy , Pregnancy, Ectopic/diagnostic imaging , UltrasonographyABSTRACT
Zona pellucida (Z.p.) thickness is believed to be considerably influenced by hormonal stimulation. Assessments of the influence of the patient's age on Z.p. thickness have been at variance. In our study we wanted to investigate, among several other parameters, the age of the patient as a possible factor influencing Z.p. thickness. Prior to intracytoplasmatic sperm injection (ICSI) we determined the mean thickness of the Z.p. of 843 metaphase-II oocytes from 100 patients, which had been obtained after ovarian stimulation in an in vitro fertilization (IVF)/ICSI program. The mean thickness of the Z.p. was significantly positively correlated with the age of the patient (p = 0.000, r = 0.129). When the intraindividual mean values of Z.p. thickness (n = 100) were used, a positive correlation with age (p = 0.174, r = 0.137) remained. However, this correlation was no longer significant. It is concluded that, beside other factors, a thicker Z.p. could be responsible for the possibly lower fertilization rate of older patients in a conventional IVF program.
Subject(s)
Cellular Senescence/physiology , Infertility, Female/physiopathology , Sperm Injections, Intracytoplasmic , Zona Pellucida/physiology , Zona Pellucida/ultrastructure , Adult , Age Factors , Cell Size/physiology , Estradiol/blood , Female , Fertilization in Vitro , Gonadotropins/administration & dosage , Humans , Metaphase , Oocytes/ultrastructureABSTRACT
At present, the long-term culture of ovarian tissue is problematic. The aim of this study was to measure apoptosis in long-term cultures of human ovarian tissue. Biopsies of human ovaries were cultured for 6 weeks. Samples were taken weekly for histological investigation. The apoptotic cells were marked with anti-caspase 3. Simultaneous to this experiment, other tissue samples were preincubated for 3 h with 1 micromol staurosporine l(-1), an inducer of apoptosis, and apoptosis was compared among samples. Furthermore, the proportion of lethal cells was determined weekly. After 6 weeks, 99% of the tissue samples showed an intact structure. They expanded in all directions on the floor of the multi-wells to form a monolayer. Apoptotic cells could be marked only sporadically (16.3 +/- 5.9 fluorescence (counts per 3600 microm(2))) after 6 weeks. After preincubation with staurosporine after the same period of culture, the proportion of apoptotic cells was significantly increased compared with that in untreated control samples (66.8 +/- 14.5 versus 16.3 +/- 5.9%, respectively; P < 0.05). Under the same experimental conditions, the proportion of lethal cells was 3.6 +/- 0.9, 3.9 +/- 2.1 and 5.2 +/- 1.5% for weeks 1, 3 and 6, respectively. After preincubation with 1 micromol staurosporine l(-1), the proportion of pyknotic cells after 6 weeks of culture was significantly higher (37.2 +/- 4.4%) than that in control samples (3.95 +/- 2.05%; P < 0.05). No significant increase in apoptosis in cultured human ovarian tissue after 6 weeks was observed compared with control tissues on day 1. These results indicate that under optimal culture conditions it is possible to cultivate human ovarian tissue long term. The influence of long-term culture on hormone synthesis and follicle maturity will be investigated further.