[Long-term sequelae of malignant tumors in childhood: consequences of late side-effects].

110 children with malignant diseases (leukemia excepted) who survived 5-20 years (median 9) post-therapy were followed (1996-1998). Median age during follow-up was 15 years (range 5-23). The most common malignancies were brain tumors, lymphoma, retinoblastoma and Wilm's tumor. The 174 late side-effects included endocrine disorders (19%), cognitive impairment (14%), orthopedic dysfunction (12%), alopecia (12%), dental damage (11%), psychological (8%) and neurological (8%) disturbances, and azoospermia or amenorrhea (5%). There was no cardiac or renal damage and no second malignancy. 29% of side-effects were severe. There was significant reduction in quality of life in 54 (49%), in 27 of whom it was severe enough to require psychological intervention. Treatment of brain tumor caused 98 late side-effects in 28 patients (sequelae-to-patient ratio [SPR] 3.3). Most cognitive, endocrine and neurological disorders, and most cases of alopecia, dental and psychological difficulties were in these patients. There were frequent late complications in those treated for retinoblastoma (SPR 1.8), and bone or soft tissue sarcomas (SPR 0.8). Those treated for Wilm's tumor had few side-effects (SPR 0.4). Late side effects were most frequent after radiation, reaching as high as SPR 2.4. It averaged only 0.5 in those treated with chemotherapy alone or in combination with surgery. Reduction of late side-effects in these patients requires using less toxic modalities, as long as cure rate is not compromised. When considering secondary strategies, screening for early detection of late complications would enable immediate solutions, such as hormonal replacement or providing compensating skills for post-treatment disability.

Ovarian stimulation in in vitro fertilization with or without the "long" gonadotropin-releasing hormone agonist protocol: effect on cycle duration and outcome.

OBJECTIVE: To study the correlation between stimulation duration of IVF cycles, with and without GnRH agonist (GnRH-a), and cycle outcome. DESIGN: Retrospective analysis of data. SETTING: University-affiliated IVF clinic. PATIENT(S): 998 IVF cycles in which long GnRH-a protocol was used, and 155 cycles with hMG only. INTERVENTION(S): IVF cycles. MAIN OUTCOME MEASURE(S): Cycle outcome in number of oocytes and embryos, and pregnancy rate. RESULT(S): The mean stimulation duration (+/-SD) was 9.6+/-1.7 and 6.7+/-1.0 for the GnRH-a and the hMG-only cycles, respectively (P<0.01). In the GnRH-a group, no statistically significant correlation between cycle duration and pregnancy rate was found. Interestingly, the patients treated for 9 days had the highest number of oocytes retrieved and the highest pregnancy rate. Stimulation duration was not affected by age in either protocol. GnRH-a cycles yielded a significantly higher number of oocytes and embryos compared to cycles without GnRH-a. The pregnancy rate was similar in both groups. CONCLUSION(S): Stimulation duration in the long GnRH-a protocol group was significantly longer than in the hMG-only group. Stimulation duration was not affected by age. No statistically significant correlation was found between stimulation duration and cycle outcome in the long protocol group.

Male gonadal function in survivors of childhood Hodgkin and non-Hodgkin lymphoma.

The aim of this study was to investigate the impact of therapy on long-term gonadal function of young people cured of childhood lymphomas and to assess whether a prepubertal state during the treatment protects the gonads from chemotherapy and/or radiotherapy late effects. Clinical evaluation, semen analysis, and endocrine status were studied in 20 survivors of childhood lymphomas. Five patients received Inverted Y radiotherapy, 2320 cGy (1550-4000); all 20 received chemotherapy as follows: MOPP/ABVD protocol, 9 patients; COMP protocol, 5 patients; MOPP protocol, 3 patients; other protocols, 3 patients. Semen analysis results were as follows: normal values, 4/20 patients; oligospermia, 8/20 patients; azoospermia, 8/20 patients; FSH above normal level, 10/20 patients; 4/5 who received Inverted Y irradiation were azoospermic and 1 was severely oligospermic. Treatment damage to the testis involves tubular germinal elements. Radiotherapy and chemotherapy combinations that included nitrogen mustard or cyclophosphamide were associated with high rates of oligospermia and azoospermia. MOPP/ABVD combination did not have a significant better outcome of sperm counts compared to MOPP alone. Age at chemotherapy did not correlate with the sperm count; hence a prepubertal state did not protect the gonad from the late effects of treatment.

A prospective randomized study comparing intramuscular with intravaginal natural progesterone in programmed thaw cycles.

A simple programmed thaw cycle is described, during which the endometrium is prepared with oral oestradiol, followed by a natural progesterone source. This method involves minimal blood tests and uses inexpensive medications. Originally, an i.m. progesterone-in-oil preparation was used. Although highly successful in achieving high serum progesterone concentrations, the daily injections of the oily compound were found to be problematic from the patients' perspective. To examine the possibility of replacing the injectable progesterone with a vaginal preparation we conducted a prospective randomized study of 1 year's duration, during which time 170 and 184 thawing cycles were done with injectable and vaginal progesterone respectively. Although the progesterone blood concentrations obtained with the injectable preparation were more than twice those obtained with the vaginal progesterone, the clinical pregnancy rates (defined as percentage thawing cycles with gestational sac(s)/embryo transfer as demonstrated on ultrasound, 4 weeks after embryo transfer) were similar for both groups (15.9 and 16.8% respectively). Implantation and abortion rates were also comparable. These results agree with previous reports of higher uterine progesterone concentrations after the vaginal application. We conclude that the combination of oral oestradiol and vaginal progesterone is an effective, simple and inexpensive approach for programmed thaw cycles.

High doses of gonadotrophin-releasing hormone antagonist in in-vitro fertilization cycles do not adversely affect the outcome of subsequent freeze-thaw cycles.

The clinical application of gonadotrophin-releasing hormone (GnRH) antagonists instead of GnRH agonists, to prevent spontaneous premature luteinizing hormone surge during ovarian stimulation for assisted reproduction treatment has been advocated. A recent, double-blind, dose-finding study, including six dosages of the GnRH antagonist ganirelix, in women undergoing ovarian stimulation with recombinant follicle stimulating hormone (FSH), has indicated that high doses of GnRH antagonist (1 or 2 mg once daily) are associated with a low implantation rate. This follow-up study reports on the pregnancy rate after replacement of cryopreserved embryos obtained in stimulation cycles of the above-mentioned trial. Ovarian stimulation was initiated on day 2 of the cycle, with daily injections of 150 IU recombinant FSH. Ganirelix (0.0625, 0.125, 0.25, 0.5, 1.0 or 2.0 mg) was administered once daily from stimulation day 6 onwards, up to and including the day of human chorionic gonadotrophin. Retrieved oocytes were fertilized by in-vitro fertilization (IVF) or intracytoplasmic sperm injection and a maximum of three fresh embryos was transferred. Excess embryos were frozen, and subsequently used in either natural or programmed cycles. Until June 1998, 11 ongoing pregnancies (12-16 weeks after embryo transfer) were achieved from 46 cycles in which embryos had been first frozen (23.9% per transfer). Six of these 11 patients had been treated with a high dose of ganirelix (1.0 or 2.0 mg) during the IVF cycles in which the embryos were obtained. In conclusion, our data suggest that high dosages of ganirelix do not adversely affect the potential of embryos to establish clinical pregnancy in freeze-thaw cycles.

The presence of a sponsoring embryo in a batch of poor quality thawed embryos significantly increases pregnancy and implantation rate.

OBJECTIVE: To evaluate quantitatively the effect of one good-quality (sponsoring) embryo in a batch of low-quality thawed embryos on the implantation and pregnancy rates (PR). DESIGN: Retrospective analysis of data. SETTING: Tertiary care center IVF clinic affiliated with a university medical school. PATIENT(S): Between March 1988 and April 1995, 392 IVF patients underwent a total of 440 thawing and ET cycles of 1,436 multicellular embryos. MAIN OUTCOME MEASURE(S): Implantation, clinical pregnancy, and multiple pregnancy rates. RESULT(S): In the absence of sponsoring embryos in the thawed batch of embryos, a PR of 9.8% with an implantation rate of 3.1% was achieved. In the presence of a single sponsoring embryo, the PR nearly doubled (18.2%), with a significantly higher implantation rate of 7.0%. Only singleton pregnancies were achieved in the absence of sponsoring embryos compared with 21.7% multiple pregnancies in the single sponsoring embryo group. CONCLUSION(S): The presence of a sponsoring embryo in a batch of poor quality thawed embryos is an important factor that significantly increased pregnancy and implantation rates. The optimal strategy for planning batches of multicellular frozen embryos is to include at least one sponsoring embryo in each batch when possible. We speculate that the sponsoring embryo may favorably influence the chances of low-quality embryos to undergo successful implantation.

Assisted implantation: direct intraendometrial embryo transfer.

We report herein a technique for direct intraendometrial transfer (DIET) of human embryos. In this study we evaluated whether 2-day embryos injected into the endometrial stroma can develop normally into viable pregnancies. After in vitro fertilization the embryos were injected into the endometrium of 14 women under direct visualization using a CO2-pulsed flexible hysteroscope. A total of 44 embryos were transferred, resulting in 2 clinical (14.3% per cycle) and 2 chemical pregnancies. In 1 patient, amniocentesis revealed monosomy X and the pregnancy was terminated at 18 weeks. The 2nd patient had an uneventful pregnancy and delivered a healthy baby. The results from the present study, demonstrating a low implantation rate after DIET of 2-day embryos, suggest that the endometrial stroma does not provide an optimal environment for early embryonic development. The acidifying effect of CO2 used for insufflation may also explain the low pregnancy rate after DIET. We conclude that it is possible to achieve pregnancy by DIET in humans, but presently this procedure can be considered only in cases where the implantation site needs to be precisely determined.

Undocumented embryos: do not trash them, FISH them.

Pronuclei formation is routinely assessed 16-20 h after oocyte insemination in in-vitro fertilization (IVF). Occasionally, the pronuclei disappear before this time, rendering them as 'undocumented'. Since the number of pronuclei detected is used to distinguish normal from abnormal embryos in the context of ploidy, the diploidy of undocumented embryos is questionable, and therefore they are routinely discarded. The introduction of fluorescent in-situ hybridization (FISH) technology allows the assessment of ploidy status in undocumented embryos that continue to cleave to form blostomeres. In this study, we used FISH to analyse the chromosomal status of 23 undocumented embryos obtained from 10 patients. Biopsied blastomeres were fixed and probed for five chromosomes (X, Y, 13, 18, 21). Diploidy was confirmed in 13 (57%) embryos while the remaining 10 embryos displayed various chromosomal anomalies. Six of the diploid embryos were transferred subsequently to the patients. One ongoing pregnancy was achieved following transfer of an undocumented, analysed embryo, which was already cleaved when assessed 20 h after insemination. We suggest that accelerated dismantling of the pronuclear membrane and subsequent cleavage do not necessarily indicate abnormal chromosomal content and may result in normal pregnancy. In a patient with a small number of embryos, FISH may be used to ascertain diploidy of undocumented embryos, thereby increasing the number of available embryos for transfer.

Thyroid function in early normal pregnancy: transient suppression of thyroid-stimulating hormone and stimulation of triiodothyronine.

In order to determine the effect of gestation on thyroid function in healthy subjects, we have prospectively evaluated thyroid function in pregnant individuals undergoing termination of pregnancy, and repeated the tests 2-3 months later. Venous blood was tested for human chorionic gonadotropin (hCG), thyroid-stimulating hormone (TSH), free thyroxine (FT4) and total triiodothyronine (TT3). Early pregnancy thyroid function tests showed a significant decrease (p < 0.001) in TSH and a significant increase (p < 0.001) in TT3 as compared to the nonpregnant state; FT4, however, did not change significantly. In 8 (11.2%) pregnant subjects, TT3 levels were above the normal range for nonpregnant controls. Elevated thyroid function in early pregnancy is transient, and does not usually warrant antithyroid treatment. Thus, any conclusion regarding thyroid function in early pregnancy should be based on pregnant controls rather than general population controls.

The significance of early second-trimester sonographic detection of minor fetal renal anomalies.

A study of 6350 consecutive transvaginal ultrasound examinations was performed as part of a routine fetal evaluation. Twenty-one cases (0.33 per cent) of early second-trimester sonographic detection of minor renal abnormalities (unilateral renal agenesis, pelvic kidney, and double collecting system) are presented. The sonographic diagnosis was made at 14-18 weeks of pregnancy and confirmed, in all of the 21 fetuses, postnatally or by post-mortem. A high incidence of associated fetal anomalies (24 per cent) and parental renal abnormalities (14 per cent) was demonstrated. Transvaginal sonography was found to be a useful tool for diagnosing these renal anomalies as early as 14 weeks of pregnancy. The likelihood of various associated anomalies and long-term implications on renal function raise questions concerning the prenatal management of such patients.

Pregnancy after subzonal insertion of cryopreserved spermatozoa from a patient with testicular seminoma.

The first pregnancy established after subzonal insertion of frozen-thawed sperm obtained from a patient with seminoma and severe oligoasthenospermia is reported. A total of 800,000 sperm with a poor progressive motility were recovered after thawing. Three to seven motile spermatozoa were injected into the perivitelline space of each of 10 oocytes, and seven sibling oocytes were coincubated and inseminated with 200,000 motile sperm/mL. Only 1 of the manipulated oocytes fertilized and was transferred to the uterus 65 hours after insemination at the eight-cell stage. A healthy boy weighing 3,600 g was delivered spontaneously at 38 weeks of pregnancy. This report gives hope to patients with testicular malignancy and severely impaired sperm function to maintain their reproductive potential through sperm banking and assisted fertilization techniques.

When do anomalies begin?

An anomaly in science is an observed fact that is difficult to explain in terms of the existing conceptual framework. Anomalies often point to the inadequacy of the current theory and herald a new one. It is argued here that certain scientific anomalies are recognized as anomalies only after they are given compelling explanations within a new conceptual framework. Before this recognition, the peculiar facts are taken as givens or are ignored in the old framework. Such a "retrorecognition" phenomenon reveals not only a significant feature of the process of scientific discovery but also an important aspect of human psychology.

Sperm motility index: a new parameter for human sperm evaluation.

OBJECTIVE: The study was performed to evaluate the correlation between sperm motility index, a novel parameter of semen quality, and routine semen analysis parameters by microscopic evaluation. DESIGN: Sperm motility index was measured by an electro-optical device, the Sperm Quality Analyzer (United Medical Systems Inc., Santa Ana, CA). Human semen samples covering the whole span of qualities were analyzed prospectively and simultaneously by both methods. SETTING: Samples were collected from patients referred to university hospital infertility clinics. PATIENTS, PARTICIPANTS: Nine hundred sixty-eight semen samples of 812 patients and healthy men were analyzed. MAIN OUTCOME MEASURE(S): Sperm motility index is a measurement of optical density fluctuations caused by motile cells; therefore, a positive correlation was anticipated between its values and semen motility parameters. RESULTS: Sperm motility index values demonstrated statistically significant correlation with motile cell concentration, total cell concentration, and percent motile cells. They were also shown to reliably represent semen quality assessment obtained by two experienced andrologists. CONCLUSIONS: The sperm motility index provides a reliable and objective reflection of semen motility parameters and quality.

Measurements of the refractive-index variations with temperature of a photomonomer.

The temperature dependence of the index of refraction of CIBATOOL XB5081, a commercial photomonomer commonly used for stereolithographic part building, is measured at wavelengths of 632.8, 514.5, and 488.0 nm.

The effect of prefreeze in vitro culturing on the success of embryo freezing in mice.

There is currently a controversy as to whether the prolonged in vitro culturing of embryos before freezing has a deleterious effect on their ability to survive freezing and thawing. We compared the survival rate of frozen/thawed mouse embryos after in vitro culturing, from the two-cell stage through the eight-cell, morula, and blastocyst stages, with the survival of embryos developed in vivo to the same stages. Following induced superovulation and mating, embryos in the desired cleavage stage were flushed from the oviducts and/or uterus and either cultured in vitro or frozen immediately in sterile glass ampoules to -40 degrees C and plunged into liquid nitrogen for storage. Dimethyl sulfoxide (1.5 M) was used as cryoprotectant. After thawing, the survival rate (determined by the morphological appearance of the embryos) was significantly lower in the eight-cell stage embryos in the group grown in vivo (P less than 0.05). The number of embryos developing into expanded and hatched blastocysts was not significantly different when the in vivo vs in vitro cultures were compared over each of the three cleavage stages: eight cells (82 vs 83%), morula (92 vs 87%), and blastocyst (33 vs 51%), respectively. There was a significant decrease in the development rate of blastocyst-stage embryos when compared with earlier stages under both culture conditions (P less than 0.001). It is concluded that, compared to in vivo-grown embryos frozen at the same stages, prolonged in vitro culture does not reduce the embryos' ability to develop normally.

Immunohistochemical localization of renin and angiotensin II in human ovaries.

Ovaries from six women with normal menstrual cycles, a follicle wall biopsy specimen from a gonadotropin-stimulated preovulatory ovary, and a corpus luteum of pregnancy were examined by immunohistochemistry for the presence of immunoreactive renin and angiotensin II. Both antisera densely stained thecal and stromal cells (interstitial complex) and luteal cells. Whereas granulosa cells in developing follicles were either unstained or lightly stained, the heavily luteinized granulosa cells of the preovulatory stimulated follicle were strongly positive for immunoreactive renin and angiotensin II. These anatomic findings are consistent with gonadotropin-stimulated local production of both renin and angiotensin II in the human ovary and support the functional roles proposed for the ovarian renin-angiotensin system in follicle development, ovulation, and luteal function and during pregnancy.

Morphologic and functional studies of immature rat oocyte-cumulus complexes after cryopreservation.

The effects of freezing and thawing (F/T) on functional activity of immature rat oocyte-cumulus complexes (OCC) were studied. The OCC were divided into three groups according to the number of cumulus-cell layers surrounding them. The OCC were then frozen and thawed (F/T), with dimethyl sulfoxide (DMSO) as cryoprotectant. The survival rates after thawing increased significantly (P less than 0.001) as the number of cumulus-cell layers increased. Germinal vesicle breakdown (GVBD) was evaluated in F/T oocytes. After 2 hours, significantly (P less than 0.05) fewer oocytes demonstrated GVBD than did those in the control group. There was no difference, however, after 4 hours of culture. A significant (P less than 0.05) decrease in follicle-stimulating hormone (FSH)-dependent cyclic adenosine monophosphate (cAMP) accumulation was observed in the F/T group. However, the amount of cAMP produced was sufficient to maintain the oocyte in meiotic arrest. There was a borderline significant decrease of the coupling between cumulus cells and the oocyte in F/T OCC, as evaluated by the transport of 3H-uridine into the oocyte. It is concluded that immature rat oocytes can be successfully cryopreserved when they are surrounded by five or more layers of cumulus cells. FSH responsiveness and intercellular communication were essentially maintained. There was a slight delay in GVBD, which needs further clarification.

Immunocytochemical localization of angiotensin II immunoreactivity and demonstration of angiotensin II binding in the rat ovary.

The cellular localization of angiotensin II immunoreactivity and the presence of angiotensin II binding in rat ovaries were studied. Angiotensin II immunohistochemical staining was demonstrated throughout the corpora lutea of gonadotropin-stimulated immature rats and pseudopregnant adult rats, as well as in some stromal and thecal cells surrounding large antral follicles. No immunostaining was observed in granulosa cells of preantral or antral rat follicles or in ovaries from unstimulated immature rats. With in vitro autoradiography, specific, saralasin-suppressible 125I-angiotensin II binding was demonstrated in normal cycling rat ovaries: diestrus greater than proestrus greater than estrus. The combined findings of angiotensin II immunostaining in ovarian follicles and corpora lutea and of cycle-related angiotensin II binding support the hypothesis of a functional role for the ovarian renin-angiotensin system.

Follicular development is impaired by inhibitors of serine proteases in the rat.

Serine proteases such as plasminogen activators are produced by granulosa cells both in vivo and in vitro and have been implicated in the process of ovulation. For a study of potential roles of serine proteases in early follicular development, immature rats were injected with pregnant mare serum gonadotropin, followed 2 hours later by laparotomy and injection of the serine protease inhibitors benzamidine and epsilon-aminocaproic acid into the bursa of one ovary. As a control, saline solution was injected into the contralateral bursa. Animals were put to death 48 hours after injection of serine protease inhibitors, and three to five randomly selected longitudinal sections were evaluated by computerized morphometry. The area occupied by antral follicles relative to the total cross-sectional area of each section was computed. Resultant ratios from serine protease inhibitor-treated ovaries were compared with those from contralateral control ovaries. Ninety-three percent of serine protease inhibitor-treated ovaries showed a reduction in antral follicular size when compared with corresponding control ovaries, which is indicative of inhibitory effects of serine protease inhibitor treatment on folliculogenesis. To further investigate this effect, ovulation was induced by human chorionic gonadotropin administration 48 hours after pregnant mare serum gonadotropin and 46 hours after serine protease inhibitor or saline solution treatment. Animals were put to death 20 hours later and the number of oocytes ovulated into oviducts was determined. Oviducts from serine protease inhibitor-treated ovaries contained 51% fewer oocytes than their control counterparts. Artifacts of surgical stress or vascular diffusion of serine protease inhibitor from treated to control sides were ruled out by appropriate control experiments. We conclude that early serine protease inhibitor treatment of pregnant mare serum gonadotropin-stimulated rat ovaries impairs folliculogenesis. Thus, in addition to involvement in ovulation, serine proteases appear to play important roles throughout follicular development.