Ultrasound and endocrine evaluation of the ovarian cycle and early pregnancy in the Sumatran rhinoceros, Dicerorhinus sumatrensis.

Longitudinal ultrasound and endocrine evaluations were conducted in two adult female Sumatran rhinoceroses (Dicerorhinus sumatrensis) over a period of 12-22 months to learn more about their reproductive physiology. Rectal ultrasonography was conducted to monitor ovarian activity. Blood samples were collected and analysed for progesterone and LH, and faecal samples were analysed for progestin metabolites. One female showed cyclic ovarian activity during the study period, whereas the other female showed no evidence of ovarian activity. The cyclic Sumatran rhinoceros appeared to be an induced ovulator, the first of its kind reported within the Perrisodactyla. Ultrasound examinations of the ovaries revealed the formation of anovulatory haemorrhagic follicles when the animal was not mated. These follicles appeared to undergo varied degrees of luteinization that resulted in irregular faecal progestin profiles. When allowed to mate, the female showed a 21 day reproductive cycle that was reflected in both faecal progestin and serum progesterone profiles. The concentration of serum LH was baseline before mating, increased approximately 30-fold within 1-2 h of intromission and returned to baseline within 22 h. Ovulation occurred within 46 h of copulation. The female conceived three times during the study. Pregnancy was detected using ultrasonography 14-16 days after mating, and the concentration of both serum progesterone and faecal progestins remained high. Early embryogenesis appeared to be similar to that in horses. However, each pregnancy terminated unexpectedly within the first 3 months of gestation. This study demonstrates the important role that basic research and reproductive technology can play in developing a natural breeding programme for an endangered animal in captivity.

Development of embryos produced by intracytoplasmic sperm injection of cat oocytes.

Development of cat oocytes following intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) was compared in two experiments. Domestic cat donors (used as a model for wild felids) were treated with 150 IU equine chorionic gonadotrophin (eCG) on treatment day 1 or a total of 10-15 IU of follicle-stimulating hormone (FSH) over four days, followed by 100 IU human chorionic gonadotrophin (hCG) on day 5 and follicular aspiration 24-26 h later. A jaguarundi (Herpailurus yaguarondi) female was stimulated twice with FSH (20 IU) or eCG (300 IU) and hCG (250 or 300 IU) before oocyte recovery. After storage at 4 degrees C, domestic cat semen was washed and processed. For ICSI, denuded oocytes were each injected with an immobilised spermatozoon. IVF oocytes were co-incubated with 5 x 10(4) motile spermatozoa/0.5 ml for 4-6 h. Noncleaving oocytes were fixed and stained 24-28 h after injection or insemination. Presumptive zygotes were cultured before transfer on day 5 (experiment I only) or evaluation on day 7 (experiments I and II). In experiment I, fertilization frequency was 67.9% (72/106) and 58.1% (122/210) for IVF and ICSI oocytes, respectively (P > 0.05). Most noncleaving ICSI oocytes (71/88, 80.7%) at 24 h were at metaphase II, of which half (35/71, 49.3%) had an activated spermatozoon (n=4) or premature chromatin condensation (PCC, n=31) of the sperm head. All 69 day 7 IVF embryos developed to morulae (> 16-cells, 46.7%) or blastocysts (53.3%), and 59/63 (93.7%) ICSI embryos reached the morula (50.8%) or blastocyst (42.9%, P > 0.05) stage. Mean cell number in IVF and ICSI embryos was 136 and 116 (P > 0.05); morulae had 77 and 46 (P < 0.05) and blastocysts had 187 and 209 (P > 0.05) cells, respectively. After transfer of 10 or 11 day 5 ICSI morulae to each of four recipients, a total of three kittens were born to two dams at 66 or 67 days. Of 18 fair-to-good quality oocytes recovered from a jaguarundi on two occasions, 10 (55.6%) embryos were produced by ICSI with fresh (n=5) or frozen (n=5) conspecific spermatozoa, but no jaguarundi kittens were born after transfer of these embryos to domestic cat recipients. In experiment II, cleavage frequency following IVF (15/17, 88.2%) and ICSI (31/38, 81.6%) was higher (P < 0.05) than following sham ICSI (13/35, 37.1%). Mean cell number (27 cells) and blastocyst development (0%) on day 7 was lower (P < 0.05) in the sham ICSI group than in the ICSI group (45 cells, 15.6% blastocysts) which, in turn, was lower (P < 0.05) than the IVF group (94 cells, 46.7% blastocysts). We have demonstrated that ICSI can be applied successfully in domestic felids and suggest that the technique will effectively augment other biotechniques being developed for enhancing reproduction in endangered felids.

In vitro and in vivo development of embryos produced by in vitro maturation and in vitro fertilization of cat oocytes.

Cumulus-oocyte complexes of domestic cats were classified by morphology of ooplasm (A = good, B = fair, C = poor) and cultured for 24 h in TCM 199 with gonadotrophins (eCG, FSH, hCG or FSH/hCG). More of type A oocytes (52%) underwent in vitro maturation (IVM) than of type B (41%) or type C (17%). The gonadotrophin source did not affect frequency of IVM of type A (50-53%) or type B (38-44%) oocytes, but IVM of type C oocytes in hCG or FSH/hCG (27%/19%) was about double that in eCG or FSH alone (13%/10%). After IVF, frequency of cleavage for type A (54%), B (41%) and C (26%) oocytes was similar to the IVM frequency of the equivalent type. After 7 days, development to the morula (M) stage in vitro was similar among types (47-58%); however, higher percentages of type A and B oocytes developed to blastocysts (Bl), 31% and 29%, respectively, than of type C (15%). After transfer of day 5 (n = 70) or 6 (n = 32) M and Bl to day 4 or 5 recipients in trial 1 (n = 4) and 2 (n = 3), respectively, the three recipients in trial 2 gave birth to four live kittens. Development in vitro to M of IVM/IVF embryos frozen in propanediol plus sucrose during early cleavage was similar (64-69%) to that of cohort controls (64%), but Bl formation was reduced (13-17% versus 32%). Damage to the zona pellucida after plunging into liquid nitrogen at -30 degrees C was higher (11%) than that of the embryos cooled at 10 degrees C min-1 from -30 degrees C to -150 degrees C before storage (2%).

Birth of a western lowland gorilla (Gorilla gorilla gorilla) following in vitro fertilization and embryo transfer.

A 21-year-old multiparous female exhibiting 31-41 day menstrual cycles was given hFSH (225 IU/day, Metrodin 75, from cycle day 3 through 9 (menses = day 1) and hCG (10,000 IU, Profasi, on day 10 to stimulate follicular development. At 35 h after hCG, under isoflurane (AErrane) anesthesia, follicles were aspirated by controlled suction under transvaginal ultrasound guidance. Metaphase II oocytes (n = 11) were placed in modified human tubal fluid (mHTF, 100 microliters) medium under oil at 37 degrees C in humidified 5% CO2. Frozen semen, collected by voluntary ejaculation, was thawed (70 degrees C H2O bath, 6 sec), diluted slowly, centrifuged, and resuspended in mHTF, and 160,000 motile spermatozoa/ml were added at 6 h after oocyte recovery. At 21 h postinsemination (p.i.) eight oocytes were at the two-cell stage, five were cryopreserved, and three were cultured to the six- to eight-cell stage in mHTF with granulosa cells before transcervical uterine transfer at 47 h p.i. using a Teflon catheter. Micronized progesterone (400 mg/d) was orally administered for 10 weeks posttransfer (p.t.). Ultrasound examination revealed a single fetus at 15 weeks p.t., and unassisted delivery of a live 1.37 kg female infant occurred at 29 weeks. Am. J. Primatol. 41:247-260, 1997.

False positive malignant peritoneal cytology and psammoma bodies in benign gynecologic disease.

OBJECTIVE: To determine the incidence of false positive malignant peritoneal cytology and psammoma bodies associated with benign gynecologic disorders. STUDY DESIGN: Pelvic peritoneal fluid was prospectively collected for cytologic examination from 119 women undergoing laparoscopy for benign conditions (infertility, 67; pelvic pain, 35; elective sterilization, 17). The median age was 30. No patient had laparoscopic gross cancer or histologic evidence of cancer. RESULTS: The peritoneal cytology of 2 of 119 cases (2%) contained cells with features suggestive of malignancy, and 6 of 119 cases (5%) contained psammoma bodies. Both cases of false positive peritoneal cytology and four of six with psammoma bodies were associated with endometriosis. All cases were followed for a minimum of two years, and no patient had developed cancer. CONCLUSION: Peritoneal fluid from cases of benign gynecologic disorders, especially endometriosis, can contain psammoma bodies and cells suggestive of malignancy.

Successful in vitro and in vivo development of in vitro fertilized two- to four-cell cat embryos following cryopreservation, culture and transfer.

In vitro and in vivo survival of in vitro-derived 2- to 4-cell cat embryos following cryopreservation was examined. Prefreeze 1- vs 2-step cryoprotectant exposure (Experiment 1) and warming method (Experiment 2) on zona pellucida damage and development in vitro were compared. To determine viability in vivo, frozen/thawed embryos were cultured in vitro to the morula/early blastocyst stage and transferred to synchronous recipients (Experiment 3). At 24 to 26 h after IVF, embryos were cryopreserved in 1.4 M propanediol (Pr)+0.125 M sucrose (Su) by cooling at 0.3 degrees C/min from -6 degrees C to -30 degrees C and storing in liquid nitrogen. Autologous embryos were cultured in vitro for 7 d. After warming for 5 sec in air and 10 sec at 37 degrees C in water (Experiments 1 to 3), or at room temperature air (22 degrees C; Experiment 2), the cryoprotectant was removed and embryos were cultured in vitro for 6 d (Experiments 1 and 2). Development was assessed after staining by counting cell numbers/embryo and determining the percentages at the 2- to 4-cell (nonsurvivor), pre (5 to 15), early (16 to 32), mid (33 to 50), late (>50) morula or blastocyst stages. Post-thaw development to late morula/blastocyst after 1-step exposure (68%, 15 min Pr+Su) was higher (P<0.05) than that after 2-step exposure (36%, 15 min Pr and 15 min Pr+Su). Both warming methods produced similar percentages of embryos with damaged zonae (13 to 15%) and equivalent development to morula/blastocyst (64 to 69%). Development in vitro to early morula/blastocyst of frozen embryos with intact zonae was similar to that of nonfrozen embryos. Following cryopreservation, most 2- to 4-cell cat embryos retained their capability for in vitro development to morula/blastocyst, and in vivo viability was demonstrated by the birth of 3 live kittens to 2 of 4 recipients following the transfer of 58 embryos.

Massive ovarian edema in a woman receiving clomiphene citrate. A case report.

There have been 65 previously reported cases of massive ovarian edema. We present a case in a woman receiving clomiphene citrate. It is an uncommon, benign condition and usually involves the right ovary. Patients frequently present with intermittent abdominal pain, menstrual irregularities and occasional virilization. Incomplete ovarian torsion and edema formation secondary to a preexisting ovarian lesion are the postulated mechanisms. Our report is unusual because it is the first of massive ovarian edema in a woman receiving clomiphene citrate.

Abnormal uterine bleeding. Finding and treating the cause.

Abnormal vaginal bleeding is a common presenting symptom with an extensive list of potential causes. In evaluating abnormal bleeding, nonuterine causes and pregnancy should first be excluded; after that, it is important to differentiate between ovulatory and anovulatory bleeding (dysfunctional uterine bleeding). Initial laboratory tests, in addition to a sensitive pregnancy test, include hemoglobin and hematocrit determinations, platelet count, and occasionally coagulation studies. An endometrial biopsy or dilation and curettage may be necessary to rule out endometrial hyperplasia or other pathology associated with the long-term unopposed estrogen stimulation experienced by anovulatory patients. In evaluating abnormal ovulatory bleeding, hysteroscopy with selected biopsy or curettage is superior to routine dilation and curettage. Anovulatory patients should be treated with an oral progestin, an oral contraceptive, or, if pregnancy is desired, an ovulation stimulant. Treatment options for patients having ovulatory cycles include an oral contraceptive, an oral progestin, danazol (Danocrine), a gonadotropin-releasing hormone agonist, mefenamic acid (Ponstel), and surgery.

Amenorrhea and chronic anovulation. Finding and addressing the underlying cause.

Amenorrhea is a common problem with a complex etiology. However, the same diagnostic approach can be used in evaluation of all amenorrheic patients. Complete history taking and physical examination are important for determining the presence of congenital abnormalities and chronic conditions. Pregnancy is the most common cause of amenorrhea and should be ruled out in initial laboratory evaluation. Progesterone can then be given to assess endogenous estrogen production and the functioning of the uterus and vagina. Response to this hormone suggests the presence of ovarian, hypothalamic, or pituitary dysfunction, which can be confirmed with further testing. Lack of response to progesterone indicates hypoestrogenism or obstruction of the outflow tract. An estrogen-progesterone challenge helps differentiate these conditions. Chronic anovulation is the second most common cause of amenorrhea. Treatment of chronic anovulation is important to prevent the effects of unopposed estrogen stimulation of the endometrium and to reduce the risk of endometrial adenocarcinoma.

The role of measurement of progestagen-associated endometrial protein in predicting adequate endometrial differentiation.

Late secretory endometrium synthesizes and secretes progestagen-associated endometrial protein (PEP), which is measurable in the serum of cycling women, and has been shown to increase in concentration during the luteal phase of the normal menstrual cycle. The purpose of this study was to determine the utility of serum PEP as a predictor of normal or inadequate luteal phase endometrial differentiation. One-hundred-and-twenty-five endometrial biopsies were taken within 4 days of a subsequent menstrual period, during the course of evaluation of infertility or recurrent abortion. Twenty-one of these biopsies demonstrated glandular/stromal asynchrony. Thirteen patients exhibited 'out-of-phase' endometrial biopsies and 91 patients had 'in-phase' biopsies. Mean PEP values significantly increased from the mid-luteal phase to the late luteal phase. Mean PEP values for patients with in-phase biopsies (43.92 units/ml) were not significantly different from those with out-of-phase biopsies (25.24 units/ml, P = 0.0942). PEP values for patients with asynchronous biopsies were intermediate. Use of clomiphene citrate did not affect these results.

Genital outflow tract obstruction in an adolescent with cloacal exstrophy.

The combination of congenital anomalies in patients with cloacal exstrophy presents a difficult management problem. Experience with these problems is limited because of the infrequent occurrence of the disorder and because few patients survive to adulthood. The present paper describes a 14-year-old female who presented with hyperchloremic metabolic acidosis, secondary to retained urine in a redundant ileal conduit, and symptoms of müllerian outflow tract obstruction. The literature is reviewed on genetic females with cloacal exstrophy, revealing a high incidence of müllerian fusion defects. Data are presented showing that women with cloacal exstrophy have a high potential incidence of genital outflow tract obstruction. These patients must be followed closely during adolescence so that diagnosis and treatment may be initiated early.

Sperm washing and intrauterine insemination for cervical factor, oligospermia, immunologic infertility and unexplained infertility.

The cumulative pregnancy rate was evaluated for 56 couples undergoing 227 intrauterine inseminations (IUIs) after sperm washing. The indications for IUI were cervical factor, oligospermia, immunologic infertility and unexplained infertility. The overall pregnancy rate was 21.4%, with a 0.289 cumulative probability of conception after six cycles. Within each category the pregnancy rates were 25% for cervical factor, 60% for women with antisperm antibodies, 20% for men with antisperm antibodies and 7% for oligospermia.

Immunohistochemical identification of prolactin and 24K protein in secretory endometrium.

Inadequate endometrial differentiation is a cause of infertility and recurrent pregnancy loss. Diagnostic histologic dating criteria may be supplemented by the immunohistochemical identification of protein markers in late secretory endometrium. Late secretory endometrium has been shown to contain prolactin. The authors report the immunohistochemical localization of prolactin and 24K protein in late secretory endometrium using monoclonal antisera and the dinitrophenyl hapten sandwich-staining technique. The appearance of these proteins in decidualized stromal cells of late secretory endometrium may provide a more specific indicator of endometrial development and differentiation, if their late appearance or absence can be correlated with pregnancy wastage.

Serum hormones and their receptors in women with normal and inadequate corpus luteum function.

The purpose of this study was to attempt to characterize women with a luteal phase defect (LPD) by cytosol estrogen and progesterone (P) levels and peroxidase activity in endometrial biopsy samples. These values were correlated with circulating serum estradiol, P, testosterone, and dehydroepiandrosterone sulfate measurements. Similar data were obtained from women during the proliferative phase of the cycle. Midluteal phase P values were significantly greater in women with normal luteal function than in those with LPD; however, some overlap of values existed. Furthermore, because no significant differences between this group and normal women were seen, we were unable to characterize women with LPD by estrogen or P receptor levels or endometrial peroxidase activity.

Dysmenorrhea in unicornis with rudimentary uterine cavity.

Two adolescents with a rare müllerian anomaly, uterus unicornis with a noncommunicating rudimentary uterine cavity, are presented because of clinical manifestations characterized by dysmenorrhea and pelvic pain requiring multiple hospitalizations and surgical procedures prior to the correct diagnosis. A high index of suspicion and proper diagnostic evaluation are essential for all adolescent patients presenting with dysmenorrhea associated with menarche and subsequent menstruation. The details of 2 cases and their diagnostic and therapeutic emphasis are presented.