Hierarchical radial and polar organisation of chromosomes in human sperm.

It is well established that chromosomes occupy distinct positions within the interphase nuclei, conferring a potential functional implication to the genome. In addition, alterations in the nuclear organisation patterns have been associated with disease phenotypes (e.g. cancer or laminopathies). The human sperm is the smallest cell in the body with specific DNA packaging and the mission of delivering the paternal genome to the oocyte during fertilisation. Studies of nuclear organisation in the sperm have postulated nonrandom chromosome position and have proposed a chromocentre model with the centromeres facing toward the interior and the telomeres toward the periphery of the nucleus. Most studies have assessed the nuclear address in the sperm longitudinally predominantly using centromeric or telomeric probes and to a lesser extent with whole chromosome paints. To date, studies investigating the radial organisation of human sperm have been limited. The purpose of this study was to utilise whole chromosome paints for six clinically important chromosomes (18, 19, 21, 22, X, and Y) to investigate nuclear address by assessing their radial and longitudinal nuclear organisation. A total of 10,800 sperm were analysed in nine normozoospermic individuals. The results have shown nonrandom chromosome position for all chromosomes using both methods of analysis. We present novel radial and polar analysis of chromosome territory localization within the human sperm nucleus. Specifically, a hierarchical organisation was observed radially with chromosomes organised from the interior to the periphery (chromosomes 22, 21, Y, X, 19, and 18 respectively) and polar organisation from the sperm head to tail (chromosomes X, 19, Y, 22, 21, and 18, respectively). We provide evidence of defined nuclear organisation in the human sperm and discuss the function of organisation and potential possible clinical ramifications of these results in regards to male infertility and early human development.

Compartmental ovarian steroidogenesis in polycystic ovary syndrome.

Compartmental ovarian steroidogenesis in vitro was investigated in polycystic ovary syndrome. Basal estrogen secretion by granulosa cells ranged from 60 to 284 pg/micrograms cell protein for 24 hours and progesterone secretion from 24 to 1646 pg/micrograms cell protein for 24 hours. In three of four specimens, the addition of either 10(-5)M testosterone or androstenedione significantly increased estrogen production, demonstrating the presence of aromatase activity. Treatment with human follicle-stimulating hormone (100 ng/mL) or human chorionic gonadotropin (100 ng/mL) significantly increased the progesterone production in three of four specimens. The thecal compartment of every patient secreted significantly more testosterone and androstenedione than the capsule and stroma and more estrogen in tissue from two of the four women. The androgen/estrogen ratio was significantly greater for the theca (16.9) than the capsule (1.1) or stroma (1.7). These data demonstrate that in polycystic ovary syndrome a portion of the follicles possess the qualitative characteristics of developing follicles, granulosa cell aromatase activity and gonadotropin responsiveness, and that the theca is likely the principal site of ovarian androgen synthesis. These findings suggest that the small follicles characteristic of polycystic ovary syndrome consist of a mixed population of developing and atretic follicles and that the peripheral androgen excess is attributable to the large mass of the thecal compartment from both follicle populations.

Menopausal hormone replacement therapy with continuous daily oral micronized estradiol and progesterone.

The safety and efficacy of a daily combination of micronized estradiol (E2) (0.7-1.05 mg) and progesterone (200-300 mg) were evaluated in ten menopausal women with moderate to severe vasomotor symptoms and/or vaginal atrophy over a 12-month study interval. For comparison, five similar women were placed on conjugated estrogens, 0.625 mg daily, and medroxyprogesterone acetate, 10 mg daily, for the first 10 days of each calendar month for 12 months. Patients were evaluated at 0, 1, 3, 6, and 12 months. Estrogens rose significantly from baseline in both groups (P less than .01). Progesterone increased significantly above baseline in the E2 and progesterone group (P less than .01), but did not change in the conjugated estrogens and medroxyprogesterone acetate users. All women on E2 and progesterone had a decrease in total cholesterol and an increase in high-density lipoprotein cholesterol from baseline (P less than .01). Those on conjugated estrogens and medroxyprogesterone acetate had no significant change from baseline in total cholesterol; however, they did have an increase in high-density lipoprotein cholesterol values (P less than .01). In the E2 and progesterone group, the endometrial histology became completely quiescent and there was no uterine bleeding after 6 months of observation. Four of five women on conjugated estrogens and medroxyprogesterone acetate continued regular withdrawal bleeding throughout the study period, but no endometrial hyperplasia was encountered. This study demonstrates that the daily administration of a combination of micronized E2 and progesterone results in symptomatic improvement, minimal side effects, an improved lipid profile, and amenorrhea without endometrial proliferation or hyperplasia in menopausal women.

Bioavailability of oral micronized progesterone.

Progesterone (P) has not been administered orally because of reportedly poor bioavailability and a rapid clearance rate. Unfortunately, the synthetic derivatives, although orally active, have a number of disadvantages and fail to mimic natural P completely. To investigate the bioavailability and short-term toxicity of oral micronized P, a standardized dose of 200 mg of micronized P was administered to nine healthy postmenopausal women and one male subject. Serial determinations of serum P concentrations demonstrated rapid absorption of P. Peak concentrations of P rose from a negligible baseline level to 17.0 +/- 4.9 ng/ml at an average of 2.8 +/- 0.35 hours after administration. The peak concentrations of P were equivalent to those observed in the midluteal phase in normal control cycles (14.1 +/- 2.7 ng/ml). All subjects exhibited significant elevation of P over baseline levels that persisted for at least 6 hours after the single oral dose and returned to initial levels by 24 hours. There was no significant change in estradiol, follicle-stimulating hormone, luteinizing hormone, cortisol, aldosterone, lipids, or hepatic enzymes during the 24-hour study interval.

Clinical application of nonselective assisted hatching of human embryos.

OBJECTIVE: To evaluate the effect of nonselective assisted hatching on pregnancy rate (PR) and to provide an alternative and simplified method for clinical application of assisted hatching. DESIGN: Retrospective analysis of clinical data. SETTING: Private infertility practice. PATIENT(S): Women from 258 consecutive stimulated IVF cycles. INTERVENTION(S): Assisted hatching was performed on each transferred embryo regardless of patient history, embryo morphology, or other selection criteria routinely applied to many IVF programs. MAIN OUTCOME MEASURE(S): Pregnancy, live birth, and implantation rates. RESULT(S): Of 258 consecutive patients who had nonselective assisted hatching, 109 (42%) had clinical pregnancies, with 93 (36%) live births and 178 (20%) embryos implanted. CONCLUSION(S): Nonselective assisted hatching resulted in an acceptable PR and provided an alternative and simplified method for clinical application of assisted hatching.

A comparison of the CO2 laser and electrocautery on postoperative intraperitoneal adhesion formation in rabbits.

The effect of the CO2 laser and microelectrocautery on postoperative intraperitoneal adhesions was examined in 31 female rabbits. Standard injuries were performed and/or repaired with each technique on the uterine peritoneum, the right uterine horn, and the left ovary. The animals were sacrificed 5 weeks later, and the adhesions were graded at each site. No significant differences were found between the two techniques for each site or all sites collectively (P greater than 0.05). The ovary had more extensive adhesions than the other soft tissue site (P less than 0.01) with both techniques. The present study suggests that there is no apparent difference between CO2 laser and conventional microelectrocautery on adhesion formation, and that the ovary seems particularly prone to adhesion formation with either technique.

The role of laparoscopic adhesiolysis in an in vitro fertilization program.

Adhesiolysis was evaluated in 82 women who had 129 laparoscopies for in vitro fertilization and embryo transfer (IVF-ET). Fifty-five women (67%) had lysis during the first laparoscopic attempt, and 20 of the 129 laparoscopies (16%) required lysis for identification and aspiration of follicles. In 11 women who had a second laparoscopy, 69% of the ovaries had improved access. With the use of laparoscopic lysis, only 3 of 82 women (4%) had insufficient ovarian access, preventing recovery of at least one oocyte. The findings support the efficacy of laparoscopic adhesiolysis and indicate that it has an important role in a program of IVF-ET in assuring initial access for oocyte recovery and in improving access for subsequent attempts.

Ovarian surgery in an infertility patient as an indication for a short-interval second-look laparoscopy: a preliminary study.

The usefulness of a short-interval second-look laparoscopy (SLL) after ovarian surgery was examined. In 23 infertile women who had either bilateral wedge resection (BWR) of polycystic ovaries, an oophorocystectomy (OC), or resection of endometriomata (RE), adhesions were graded before and after surgery and before and after laparoscopic lysis for each ovary. At SLL, periovarian adhesions involving at least one ovary were observed in all women. Of the 22 women who wanted to become pregnant, 3 of 3 with BWR, 1 of 3 with OC, and 6 of 16 with RE conceived. In the women with residual adhesive disease in both or the only remaining adnexa after SLL, there were no pregnancies; whereas 67% of the women with at least one adnexa free of disease became pregnant (P = 0.005). This preliminary study indicates that ovarian surgery does result in significant adhesion formation and that SLL offers an opportunity to reassess the pelvis, to lyse adhesions, and to establish a postoperative prognosis.

Hyperprolactinemic response after bromocriptine withdrawal in women with prolactin-secreting pituitary tumors.

In patients with prolactin (PRL)-secreting tumors, plasma PRL concentrations after discontinuation of bromocriptine therapy have been used clinically as an index of tumor activity. To investigate the pattern of PRL response under these conditions, seven women on chronic bromocriptine therapy for PRL-producing pituitary tumors were followed with serial PRL determinations for 2 months after bromocriptine withdrawal. In these patients, peak PRL concentrations were achieved 28.1 days (mean; range, 14 to 49 days) after bromocriptine discontinuation. However, PRL concentrations did not usually plateau until at least 40 days after cessation of therapy, and the pattern of PRL response was highly variable. Because of the inconsistent pattern of serum PRL response during the first 40 days after discontinuation of bromocriptine, decisions regarding tumor activity based on PRL concentrations should not be made until at least 6 weeks after withdrawal of the drug.

Spironolactone in combination drug therapy for unresponsive hirsutism.

Clinical and laboratory evaluations of nine hirsute women were performed for determination the efficacy of combination drug therapy. Each patient had previously failed to respond to single drug therapy with oral contraceptives (OC), dexamethasone (DEX), or spironolactone (S) and received S (100 to 150 mg) and either an OC (mestranol, 0.05 to 0.08 mg, and norethindrone, 1 mg) or DEX (0.5 mg) daily. Total testosterone, dehydroepiandrosterone sulfate, free testosterone, and sex-hormone-binding globulin were measured before therapy and 4 to 6 weeks after initiation of therapy and were compared with the responses to OC (n = 7), DEX (n = 8), and S (n = 6). A satisfactory clinical response in the rate of hair growth was defined as at least a doubling of the time interval between adjunctive therapies (electrolysis, shaving, or bleaching) and patient satisfaction with treatment. The responses of the androgenic parameters were not statistically different between combination and single drug therapy. Although all patients noted a subjective improvement in hair growth, eight of nine fulfilled the criteria for a clinical response (P less than 0.001). Transient diuresis was the only side effect noted. The study suggests that combination drug therapy is an efficacious and well-tolerated approach to the management of unresponsive hirsutism.

Cycle of conception endometrial biopsy.

Although controversial, the diagnosis of luteal phase inadequacy and its therapy may improve reproductive outcome, but an endometrial biopsy in the cycle of conception (COC) might theoretically interrupt an intrauterine pregnancy. Fifty-four biopsies obtained in the COC were identified, and patient outcome was documented. Eleven (20%) of the 54 women who underwent COC biopsy did not deliver viable infants. Two patients had ectopic pregnancies, and nine had early abortions, including one whose biopsy specimen contained an early implantation site and another with a trisomy 16 fetus. Although COC endometrial biopsy did not appear to increase the incidence of fetal wastage, biopsy information provided no predictive information suggestive of ultimate pregnancy outcome. Because no useful information is gained from a COC biopsy, we recommend either that pregnancy be avoided or a sensitive pregnancy test be employed for detection in a cycle in which a biopsy is to be performed.

Comparison of two brands of clomiphene citrate for stimulation of follicular development in a program for in vitro fertilization.

Although clomiphene citrate (CC) is used frequently to stimulate multiple follicular development in in vitro fertilization programs, comparison of the two commercially available types has been limited. Therefore, a comparison was made of Serophene (Serono Laboratories, Inc., Randolph, MA) and Clomid (Merrell Dow Pharmaceuticals, Inc., Cincinnati, OH), using the same dosage regimen of CC (150 mg/day for days 3 to 7 of the cycle). Weights and ages of the two groups were not different. Serum estradiol (E2) levels were significantly elevated in Clomid cycles, but the number of developing follicles was not different between the two brands. The percentages of patients in each group who underwent laparoscopy, oocyte recovery, oocyte fertilization, and embryo transfer were also equivalent. Among patients undergoing laparoscopy, no differences in oocyte recovery, oocyte fertilization, and embryo development were noted. Thus, despite greater elevation of serum E2 levels with Clomid, no difference in stimulation regimen outcome was observed.

The problem of polyspermy in in vitro fertilization.

Polyspermy is a potential complication of attempts at in vitro fertilization. Nine polyspermic oocytes were identified among 169 oocytes obtained from 67 cycles stimulated by human menopausal gonadotropin. Cleavage to the 2-, 3-, and 4-cell stage was observed. Four polyspermic oocytes were identified among 85 oocytes obtained from 47 cycles stimulated by clomiphene citrate. Cleavage was not observed. Careful dispersion of cumulus cells at 15 to 18 hours and examination of the oocytes for polyspermy is essential, because the condition may not be apparent at 40 hours from insemination, when normal-appearing cleavage stages may be observed.

Clinical application of intracytoplasmic sperm injection using in vitro cultured testicular spermatozoa obtained the day before egg retrieval.

OBJECTIVE: To study the effect of in vitro culture on the quality of human testicular sperm and the efficiency of intracytoplasmic sperm injection with in vitro cultured testicular sperm. DESIGN: Clinical study. SETTING: A private IVF center. PATIENT(S): Twenty consecutively seen IVF patients undergoing testicular biopsies for ICSI. INTERVENTION(S): The testicular specimens were cultured in vitro for 24 hours and the isolated spermatozoa were microinjected. MAIN OUTCOME MEASURE(S): Preincubation and postculture sperm motility, and fertilization, implantation, and pregnancy rates after intracytoplasmic sperm injection. RESULT(S): Motility increased from initial nonmotile or twitching sperm to free motile sperm in 18 of 20 cases. The injection of in vitro cultured testicular sperm resulted in a fertilization rate of 58%, an implantation rate of 20%, and a pregnancy rate of 45%. CONCLUSION(S): A testicular biopsy procedure can be performed the day before egg retrieval. Despite the low initial sperm quality, a high percentage of the prepared testicular sperm showed increased motility after 24 hours of culture. The injection of in vitro cultured testicular sperm into matured oocytes resulted in fertilization, implantation, and pregnancy rates comparable to those obtained with ejaculated sperm.

The efficacy of early pregnancy monitoring with serial chorionic gonadotropin determinations and real-time sonography in an infertility population.

Ninety-one pregnancies were monitored prospectively with serial human chorionic gonadotropin (hCG) determinations and real-time sonography. The monitoring process included an initial hCG doubling time (DT) followed by sonographic examination for fetal heart motion at 7 to 8 weeks in the asymptomatic patient. In women with an abnormal DT or who developed symptoms, repeat hCG determinations and/or sonography were performed. A single DT correctly identified 95% of the successful pregnancies (58) and 64% of the abnormal pregnancies (25 abortions and 8 ectopic gestations) in asymptomatic women. With repeat hCG determinations and/or sonography, 88% of the spontaneous abortions (before aborting) and 100% of the ectopic pregnancies (before tubal rupture) were identified. As a result of the early diagnosis, conservative surgery was performed in six of eight women with tubal pregnancies. The presence of fetal heart motion was a reliable indicator that an intrauterine pregnancy will progress to viability in both the symptomatic (89%) and asymptomatic patient (93%). We conclude that the combined use of serial hCG determinations and real-time sonography provides efficacious monitoring of the early high-risk pregnancy.

Antiestrogenic effect of clomiphene citrate: correlation with serum estradiol concentrations.

The antiestrogenic effect of clomiphene citrate (CC) on cervical mucus was evaluated in women receiving 150 mg CC daily for 5 days. Daily cervical mucus scores and serum estradiol (E2) concentrations were determined in control (n = 25) and CC (n = 24) cycles. E2 concentrations were significantly higher in the CC-treated women (mean +/- standard error of the mean, 1254 +/- 102 pg/ml versus 337 +/- 18 pg/ml, P less than 0.0001). Despite supraphysiologic E2 concentrations, however, cervical mucus scores were significantly reduced in the CC-treated group (P less than 0.01). These data indicate that CC exerts a direct suppressive effect on cervical mucus despite markedly increased E2 concentrations.

A comparison of post-thaw results between cryopreserved embryos derived from intracytoplasmic sperm injection and those from conventional IVF.

OBJECTIVE: To study the effect of freezing on early stage embryos derived from intracytoplasmic sperm injection (ICSI) or from IVF. DESIGN: Prospective, controlled clinical study. SETTING: Private IVF center. PATIENT(S): Sixty-seven consecutive patients undergoing frozen-thawed embryo transfer cycles. INTERVENTION(S): Early stage embryos were frozen, thawed, and transferred. MAIN OUTCOME MEASURE(S): Post-thaw survival, implantation and pregnancy rates. RESULT(S): We noted an 88% post-thaw survival rate, an 18% implantation rate, and a 52% pregnancy rate in the ICSI group and 81%, 11%, and 25%, respectively, with conventional fertilization. CONCLUSION(S): Early stage embryos (either zygote or 2-4 cells) derived from ICSI can be frozen with confidence and higher post-thaw survival and pregnancy rates can be achieved when compared with those from conventional IVF.

Use of a sensitive urine pregnancy test before endometrial biopsies taken in the late luteal phase.

Endometrial biopsies for evaluation of the luteal phase should be taken within 2 days of the onset of menses. When these guidelines are followed and patients present at least 12 days after the thermogenic shift on BBT, the ICON pregnancy test is extremely rapid, sensitive, specific, and has predictive values close to 100%. If a biopsy is undertaken before this time there may be an increased risk of false negative results (i.e., a decreased sensitivity). The improved technology in urine pregnancy tests has now made it feasible to obtain accurate urine pregnancy tests before endometrial instrumentation with an increased level of confidence. It is recommended that this technology be used in the management of patients undergoing endometrial biopsies in the late luteal phase.

Outcome of progesterone treatment of luteal phase inadequacy.

Diagnosis, choice of therapy, and pregnancy outcome were analyzed in 79 women evaluated for luteal phase inadequacy. Criteria for the diagnosis were established, and groups at risk for luteal inadequacy were identified. Treatment choices, tailored to the suspected cause, included progesterone suppositories in 54 women, with 23 pregnancies and 19 deliveries; clomiphene citrate in 6 women, with 2 pregnancies and deliveries; and combined treatment in 7 women, with 5 pregnancies and 4 deliveries. Eight women received no treatment, including three who underwent endometrial biopsy in the cycle of conception and who subsequently delivered. These data suggest that careful diagnosis and the proper choice of treatment are important, and that progesterone supplementation may result in improved pregnancy outcome for patients with infertility and pregnancy wastage who have luteal phase inadequacy.

Ureteral abnormalities in women with endometriosis.

High-volume intravenous urography was performed in 63 women with surgically proven endometriosis. Subtle abnormalities were found in 15.9% of these women. No patient had urologic symptoms, and there was no evidence of hydroureter or ureteral obstruction on the IVP. Long-term follow-up study will be required to determine whether or not these lesions will progress and cause ureteral obstruction.