Effect of cyclophosphamide on the immature rat ovary.

To investigate the early ovarian changes after cyclophosphamide treatment, immature rats primed for 48 h with pregnant mare serum gonadotropin were given injections i.p. of cyclophosphamide (100 mg/kg) at 1, 2, 4, 16, and 24 h before decapitation. Serum estradiol dropped significantly after 24 h of exposure to cyclophosphamide (P less than 0.001). Following 16 and 24 h of cyclophosphamide exposure, (a) the number of granulosa cells expressed from each ovary decreased (P less than 0.05 and P less than 0.01, respectively); (b) the number of nucleated bone marrow cells decreased (P less than 0.01 and P less than 0.01), and their median nuclear size was significantly reduced (P less than 0.05 and P less than 0.05) as measured by Coulter Counter and C-256 channelyzer (Hialeah, FL); and (c) the mean follicular diameter and the number of follicles with diameters greater than 300 microns were significantly lower than in control. After 4, 16, and 24 h of exposure, median granulosa cell nuclear size significantly increased (P less than 0.05, P less than 0.01, and P less than 0.01, respectively). DNA cross-links in granulosa cells, measured by alkaline elution, reached a maximum at 2 h of exposure and decreased thereafter. The above findings demonstrate that cyclophosphamide has significant effects on the rat ovary structure and function and that the granulosa cell is an important target of cyclophosphamide-induced ovarian toxicity.

A luteinizing hormone-releasing hormone agonist for the prevention of chemotherapy-induced ovarian follicular loss in rats.

In an attempt to prevent chemotherapy-induced ovarian follicular loss, [D-Leu6, des-Gly10-NH2]-luteinizing hormone-releasing hormone ethylamide (LHRHa) was given subcutaneously to Sprague-Dawley cycling female rats in two daily doses of 2.5 micrograms starting 2 days prior to and concomitant with cyclophosphamide (CTX) (5 mg/kg/day for 21 days). Four groups of female cycling rats (10 in each) received either no treatment, CTX alone, CTX + LHRHa, or LHRHa alone. One ovary from each animal was serially sectioned, stained, and examined for the number and size of follicles. CTX produced a significant reduction in the total number of follicles. The pool of growing follicles (medium to large, greater than 30 microns in diameter) appeared to be vulnerable to the cytotoxic effect of CTX. LHRHa resulted in a significant reduction in the number of medium-to-large follicles and an increase in the number of small follicles. When given in combination with CTX, LHRHa significantly further reduced the number of medium-to-large follicles, significantly increased the number of small follicles, and resulted in an increase in the total number of follicles. Chronic LHRHa treatment resulted in functional deprivation of follicles from gonadotropins, thus halting the process of recruitment from the quiescent pool of primordial follicles into the CTX sensitive pool and thereby preserving the functional potential of the ovary.

Inhibition of rat ovarian [3H]thymidine uptake by luteinizing hormone-releasing hormone agonists: a possible mechanism for preventing damage by cytotoxic agents.

To investigate the mechanism involved in luteinizing hormone-releasing hormone (LHRH) agonists' protective effects against chemotherapy-induced ovarian damage, female rats were either implanted with 1-mg pellets of LHRH agonist Zoladex (LHRHz) or sham operated. All rats were implanted with osmotic minipumps loaded with [3H]thymidine 48 h before sacrifice in diestrus. Ovaries were combusted in a biological material oxidizer. Tritiated water was recovered in a special cocktail, and ovarian tritiated thymidine uptake (3HTU) was calculated. In five experiments, LHRHz significantly reduced ovarian 3HTU. This was observed 5 days after implanting LHRHz pellets. Ovarian 3HTU correlated significantly with serum estradiol, LH, and ovarian and uterine weights. Autoradiography showed that almost all ovarian 3HTU is by granulosa cells. These data suggest that LHRHz suppresses ovarian mitotic activity. Since cytotoxic agents preferentially destroy rapidly dividing cells, our findings may represent a mechanism for ovarian protection.

Is radiation-induced ovarian failure in rhesus monkeys preventable by luteinizing hormone-releasing hormone agonists?: Preliminary observations.

With the advent of cancer therapy, increasing numbers of cancer patients are achieving long term survival. Impaired ovarian function after radiation therapy has been reported in several studies. Some investigators have suggested that luteinizing hormone-releasing hormone agonists (LHRHa) can prevent radiation-induced ovarian injury in rodents. Adult female rhesus monkeys were given either vehicle or Leuprolide acetate before, during, and after radiation. Radiation was given in a dose of 200 rads/day for a total of 4000 rads to the ovaries. Frequent serum samples were assayed for estradiol (E2) and FSH. Ovariectomy was performed later. Ovaries were processed and serially sectioned. Follicle count and size distribution were determined. Shortly after radiation started, E2 dropped to low levels, at which it remained, whereas serum FSH level, which was low before radiation, rose soon after starting radiation. In monkeys treated with a combination of LHRHa and radiation, FSH started rising soon after the LHRHa-loaded minipump was removed (after the end of radiation). Serum E2 increased after the end of LHRHa treatment in the nonirradiated monkey, but not in the irradiated monkey. Follicle counts were not preserved in the LHRHa-treated monkeys that received radiation. The data demonstrated no protective effect of LHRHa treatment against radiation-induced ovarian injury in this rhesus monkey model.

Alterations in thymic and bone marrow lymphocyte subpopulations in GnRH agonist treated prepubertal female mice.

Complex endocrine relationships exist among the hypothalamus, pituitary, ovaries and thymus. There is also considerable evidence showing gonadotropin releasing hormone (GnRH) involvement in modulating immune system functions. The present study investigated the sequential changes in functional lymphocyte subsets in primary lymphoid tissues of prepubertal female mice in vivo following GnRH agonist treatment in slow release microcapsule formulation. A direct two color immunofluorescence staining followed by flow cytometry was employed. Single i.m injection of agonist significantly decreased both absolute and relative thymic weights and absolute thymocyte counts. No differences, however, were observed in the percentage of thymocytes expressing Thy 1.2, CD4 and CD8. Absolute levels of thymic T cells, CD8 positive cells, immature cells expressing both CD4 and CD8, and immature subsets differentiating toward CD4 were significantly reduced two weeks after agonist treatment. The percentage of bone marrow B cells was also significantly decreased at the second and third weeks following agonist administration. Functional studies to determine in vivo cell-mediated immune function also indicated a significant suppression following agonist administration. These data, together with our earlier observations on secondary lymphoid tissues, suggest a general suppression of lymphocyte maturation at an early stem cell stage of development in prepubertal female mice in vivo.

New technique for selective transcervical osteal salpingography and catheterization in the diagnosis and treatment of proximal tubal obstruction.

Transcervical fallopian tube catheterization to recanalize the proximal (uterine end) fallopian tube are rapidly gaining acceptance for the diagnosis and treatment of tubal obstruction. We describe a new simplified technique for performing this procedure. Our technique obviates the need for hysterocaths and assures delivery of an atraumatic spherical tip of a cannula to the uterotubal junction. Compared with other techniques, it is quick with no need for IV or paracervical anesthesia or analgesia, no cervical dilatation, nor the use of Hyskon nor CO2 insufflation devices.

Bone density and reproductive hormones in patients with neuroleptic-induced hyperprolactinemia.

To study the prevalence of osteoporosis and hyperandrogenism in neuroleptic-induced hyperprolactinemia, the authors evaluated 10 patients. Three were amenorrheic, while seven had oligomenorrhea. Nine patients had galactorrhea. The Ferriman-Gallway hirsutism score was 12 +/- 2. Vaginal smear maturation value was 53 +/- 8. Bone density, measured by dual photon absorptionometry in the spine, femoral neck, Ward's triangle, and trochanteric region, was 98 +/- 1.5, 92.7 +/- 3, 88.5 +/- 4.2, and 92.6 +/- 3.3 percentile of controls matched for age, sex, weight, and ethnicity, respectively. Serum prolactin, follicle-stimulating hormone (FSH), luteinizing hormone (LH), dehydroepiandrosterone sulfate (DHEAS), total testosterone, and free testosterone were 82 +/- 10 ng/ml, 8.5 +/- 1.1 mIU/ml, 11.1 +/- 2.6 mIU/ml, 4695 +/- 594 ng/ml, 90 +/- 17 ng%, and 2.36% +/- 0.3%, respectively. Serum thyroid-stimulating hormone (TSH) and free thyroxine index were normal. Bone density strongly correlated with vaginal maturation value (r = 0.904, P less than 0.01). It is concluded that (1) neuroleptic-induced hyperprolactinemia is associated with hirsutism and androgen excess primarily of adrenal origin and (2) a subset of these patients is at an increased risk of developing osteoporosis. It may be possible to identify patients at risk of osteoporosis by examining vaginal smears for maturation value. Early detection and management are imperative in this group of patients.

Clinical value of prolactin bioassay in euprolactinemic reproductive disorders.

To examine the disparity between clinical presentation and prolactin (PRL) measured by radioimmunoassay (RIA), serum samples from 128 patients with galactorrhea and/or reproductive disorders were evaluated by RIA for immunoassayable PRL (RIA-PRL) and by Nb2 lymphoma cell proliferation assay for bioassayable PRL (bioassay-PRL). One hundred fifteen patients had normal RIA-PRL and 13 patients had high RIA-PRL (greater than 25 ng/ml). Twenty patients had galactorrhea, two of whom had hyperprolactinemia. The reproductive disorders in female patients included infertility, amenorrhea, oligomenorrhea, irregular menstrual cycles, and luteal phase defects. Six oligospermic males also were studied. Twenty-three male and female volunteers with no evidence of reproductive disorders served as controls. Appropriate comparisons showed that PRL bioassay/RIA ratio, an index of agreement between the two assay systems, did not differ for the various patient groups compared with controls. It is concluded that Nb2 lymphoma bioassay does not provide additional diagnostic value to RIA in defining the cause of euprolactinemic galactorrhea and/or reproductive disorders.

Chemotherapy-induced premature ovarian failure: mechanisms and prevention.

Significant advances have been made in the previously unexplored areas of the mechanisms involved in cyclophosphamide (CTX)-induced ovarian toxicity and the protective effects of luteinizing hormone-releasing hormone (LHRH agonists. The structure and function of granulosa cells and oocytes are affected by the chemotherapeutic agent, CTX. Results of experiments in female rats indicate that LHRH agonists may protect the ovaries from the toxic effects of chemotherapy. The protective effect may be related to the inhibition of ovarian mitotic activity during LHRH agonist administration. This inhibition is much more pronounced in female compared to male rats. This may be related to the observed better gonadal protective effects in females compared to males. Further experiments are underway to determine whether similar protective effects occur in female primates.

Effect of cyclophosphamide on mouse oocyte in vitro fertilization and cleavage: recovery.

To investigate the mechanism of cyclophosphamide (CTX)-induced ovarian failure, we previously reported that CTX metabolites added in vitro inhibit mouse oocyte fertilization and embryo development. In this study, we injected CTX (100 mg/kg) intraperitoneally in female mice at 0, 1, 4, 14, 18, and 24 h before sacrifice. Mice were superovulated with PMSG and hCG. Oocytes were recovered, washed, and fertilized with sperm obtained from nontreated proven breeders, and incubated for 3 days in 5% CO2 in air. CTX reduced oocyte fertilization and early cleavage rates. To examine the recovery process, CTX was injected at 0, 1, 3, 7, and 14 days before sacrifice. The most pronounced adverse effects on oocyte number and function were observed 1 and 3 days after exposure to CTX. Evidence of partial recovery was observed one week after CTX treatment. The data demonstrate that exposure of oocytes to CTX metabolites in vivo adversely effects oocyte function. This process, however, appears to be partially reversible. The oocytes may be involved in the mechanism of CTX-induced ovarian failure.

Reproductive performance of female rats treated with cyclophosphamide and/or LHRH agonist.

Previous studies addressing the ovarian protective effects of luteinizing hormone releasing hormone agonists (LHRHa) against adverse effects of chemotherapy have examined histologic and/or hormonal parameters without evaluating reproductive performance. In this report, we initially established a model for chronic treatment of female rats with cyclophosphamide (CTX) that allowed long term survival. In a second experiment, thirty seven female cycling rats (age: 70 days) were divided into 4 treatment groups. They were given either CTX (4 mg/kg/day, 5 days/week) for a total of 76 days (217 mg/kg) and/or the LHRHa leuprolide (Lupron) 5 micrograms/day by subcutaneous minipump (Alza) for 98 days. LHRHa was started 10 days before CTX and ended 12 days after the last CTX injection. All LHRHa-treated rats entered persistent diestrus. At the end of treatment, most rats treated with CTX only were in persistent estrus. Breeding was started at 218 days of age. A laparotomy to count implantation sites was performed 15 to 16 days after vaginal plug/sperm was demonstrated. All nonpregnant rats were remated. Chi square and ANOVA were used for statistical analysis. The data presented demonstrate that: 1. LHRHa given before and after CTX increased the pregnancy rate/mating (from 4/11 to 9/10; P < 0.05), the number of implantations/mated rat (from 2.5 +/- 1.4 to 13.7 +/- 1.7; P < 0.01), and reduced the need for remating (from 7/11 to 1/10; P < 0.05); 2. LHRHa-treated rats performed better than controls. We conclude that LHRHa protects against chemotherapy-induced fertility reduction in female rats.

The effect of "activated" cyclophosphamide on human and rat ovarian granulosa cells in vitro.

We investigated the mechanism of cyclophosphamide (CTX) induced ovarian toxicity by studying the effect of an activated form, 4-hydroperoxycyclophosphamide (PCTX), on human and rat granulosa cell function in vitro. In previous experiments we demonstrated that in short-term incubations with rat granulosa cells, high (greater than or equal to 100 micrograms/mL) but not low (less than or equal to 10 micrograms/mL) PCTX concentrations inhibited progesterone accumulation in vitro. In this study, human granulosa cells were obtained from patients undergoing follicle puncture for in vitro fertilization. PCTX at 10, 100, and 500 micrograms/mL, but not at 1 micrograms/mL, resulted in a dose-related reduction in progesterone accumulation in short-term human granulosa cell cultures. Rat granulosa cells were obtained from PMSG-primed immature rats and incubated for 24 h with PCTX concentrations of 1, 5, and 10 micrograms/mL. Ovine LH (10 ng/mL) was added in selected tubes. In comparison to control, progesterone accumulation was significantly reduced by PCTX concentration of 10 micrograms/mL. The above findings demonstrate that cyclophosphamide metabolites, at concentrations achievable in vivo during CTX therapy, decrease rat and human granulosa cell function in vitro. The effect of PCTX on granulosa cells is dependent on PCTX concentration and duration of exposure, as well as the species from which granulosa cells are obtained.

Effect of "activated" cyclophosphamide on mouse oocyte in vitro fertilization and cleavage.

To investigate the involvement of oocytes in the mechanism of chemotherapy-induced ovarian failure, ovulation was induced in mice using pregnant mares' serum gonadotropin and human chorionic gonadotropin. Oocytes (with cumulus) were incubated for 4 hours with "activated" cyclophosphamide, 4-hydroperoxycyclophosphamide (PCTX) at 0, 1, 10, 100, and 500 micrograms/mL. Oocytes were then washed, fertilized with sperm obtained from nontreated male proven breeders, and incubated for 4 days. "Activated" cyclophosphamide inhibited dissolution of the cumulus and reduced fertilization and early cleavage rates in a dose-related manner. The data demonstrate that exposure of oocytes to cyclophosphamide metabolites in vitro adversely affects oocyte function. Oocytes may be involved in the mechanism of cyclophosphamide-induced ovarian failure.

Effects of oocyte exposure to local anesthetics on in vitro fertilization and embryo development in the mouse.

The effect on fertilization and development of local anesthetics routinely used during ultrasound-guided oocyte retrieval in women undergoing in vitro fertilization was examined in a mouse in vitro fertilization system. Mouse oocytes were exposed in vitro to lidocaine, chloroprocaine, and bupivacaine at concentrations of 0 (control), 0.01, 0.1, 1.0, 10.0, 100.0 micrograms/mL for 30 min, washed, and then inseminated. In vitro oocyte fertilization at 24 and 48 h and embryo development at 72 h were determined. Bupivacaine adversely affected mouse in vitro fertilization and embryo development only at the highest exposure concentration, 100 micrograms/mL, while lidocaine and chloroprocaine produced adverse effects at concentrations as low as 1.0 and 0.1 microgram/mL, respectively. Furthermore, an adverse dose-related effect on fertilization and embryo development was shown for lidocaine and chloroprocaine, but not for bupivacaine. These data demonstrate that the local anesthetics, lidocaine (L), chloroprocaine (C), and bupivacaine (B), adversely affect mouse in vitro fertilization and embryo development in the order of C greater than L greater than B.

Vinblastine reduces progesterone and prostaglandin E production by rat granulosa cells in vitro.

To elucidate the possible mechanism(s) of vinblastine-induced premature ovarian failure, we studied the effect of vinblastine (VLB) on progesterone (P) and prostaglandin E (PGE) production by rat granulosa cells in vitro. Granulosa cells obtained from immature, pregnant mares' serum gonadotrophin-primed rats were incubated for 5 h in modified Medium 199 +/- LH (10 ng/mL) with varying concentrations of VLB (0.001 to 10.0 micrograms/mL) and cells plus media were assayed for total P and PGE. VLB reduced production of both basal and LH-stimulated P in granulosa cells, with VLB at a concentration of 0.1 micrograms/mL showing the first significant difference from control. This dose also suppressed basal and LH-stimulated PGE. Granulosa cell survival was similar for all groups. Thus VLB, an agent known to disrupt microtubular function, reduced granulosa cell production of both P and PGE. Our results suggest that at a concentration (0.1 micrograms/mL) lower than that routinely achieved during chemotherapy (0.3 micrograms/mL), VLB depresses rat granulosa cell function in vitro. This system appears to be a valid model for preliminary assessment of the cytotoxic effects of chemotherapy on an ovarian component.

The effect of "activated" cyclophosphamide on rat granulosa cells in vitro.

We investigated the mechanism of cyclophosphamide (CTX)-induced ovarian toxicity by studying the effect of an activated form, 4-hydroperoxycyclophosphamide (PCTX), on rat granulosa cells in vitro. Cells were obtained from PMSG-primed immature rats and incubated with PCTX at concentrations of 1, 10, 100, and 500 micrograms/mL. Ovine LH (10 ng/mL) was added in selected tubes. Cell viability before and after seven hours incubation was determined. Progesterone and prostaglandin E accumulation were measured by radioimmunoassay. Granulosa cell viability was significantly decreased at PCTX concentrations of 10 micrograms/mL or higher in a dose-related manner. PCTX at concentrations of 100 micrograms/mL and 500 micrograms/mL significantly decreased basal and LH-induced progesterone and prostaglandin E accumulation. The above findings demonstrate that cyclophosphamide metabolites decrease granulosa cell survival and function in vitro. These direct effects suggest a possible mechanism for CTX-induced premature ovarian failure.

Endometrial carcinoma presenting as a large inguinal mass.

Patients with endometrial cancer usually present with vaginal bleeding. A case of endometrial cancer presenting as an asymptomatic, large, inguinal mass is reported. This unusual clinical picture and its management are discussed.

Osteoporosis associated with neuroleptic treatment. A case report.

A 28-year-old woman developed osteoporosis following seven years of neuroleptic use. She presented with amenorrhea and profuse galactorrhea of four years' duration. Dual photon absorptionometry demonstrated reduced bone mineral density in the femur and spine. Serum calcium, phosphorus and alkaline phosphatase were normal. The patient was started on bromocriptine, and her bone density, serum prolactin, dehydroepiandrosterone sulfate, and free and total testosterone improved. No deterioration in her psychiatric condition occurred.

Postabortal paracervical abscess as a complication of paracervical block anesthesia. A case report.

A paracervical abscess occurred after paracervical block anesthesia was administered for induced abortion in an 18-year-old multigravida. She presented with vaginal, low abdominal and low back pain and with nausea, vomiting, chills and fever. Incision and drainage of the abscess were performed and a vaginal drain inserted under antibiotic coverage. Cultures of the abscess contents revealed multiple anaerobic organisms. Laparoscopy showed normal pelvic organs, and the peritoneal fluid cultures were negative. Postoperatively the patient became afebrile and was discharged after three days on antibiotics. To our knowledge, this case report is the first one on paracervical abscess as a complication of induced abortion with paracervical block anesthesia.

Urethral stones in women. A case report.

Urethral stones are encountered rarely in the urethra in women. We report the clinical and urodynamic findings and the management of 17 stones in a urethral diverticulum.