Factors related to successful ovulation induction in patients with WHO group II anovulatory infertility.

To identify baseline characteristics related to successful ovulation induction, data were analysed from oligo- or anovulatory patients undergoing their first cycle of human recombinant FSH (r-hFSH; follitropin alfa) in a chronic low-dose (75 IU starting dose), step-up protocol in two clinical trials (n=446). Patients were grouped according to response: group A, ovulated within 14 days (75 IU follitropin alfa); group B, ovulated after 14 days (>75 IU follitropin alfa); group C, not administered human chorionic gonadotrophin (HCG) because of poor response; group D, cycle cancelled due to over-response (HCG not administered); group E, spontaneous ovulation prior to obtaining criteria for administration of HCG. Mean body mass index (BMI) of group A (25.0 kg/m(2)) was significantly lower than groups B (27.1 kg/m(2), P<0.001) or C (28.2 kg/m(2), P<0.0001), but similar to group D (24.3 kg/m(2)). Mean antral follicle count (AFC) of group A was also significantly lower than group C (18.3 versus 22.7; P=0.018), but not significantly different from groups B (21.5) or D (19.5); group E had the highest mean AFC (35.7). Comparatively low BMI, low AFC and higher (although still within the normal range) FSH concentration at baseline were associated with successful ovulation induction in infertile women undergoing a chronic low-dose, step-up stimulation protocol.

Sperm as a noninvasive gene delivery system for preimplantation embryos.

OBJECTIVE: To determine if sperm could be manipulated to be a noninvasive transport carrier for the delivery of gene fragments to the blastocyst. DESIGN: Sperm cells carrying foreign DNA fragments from human papillomavirus (HPV) types 16, 18, 31, and 33 were allowed to migrate from one end of an artificial reproductive tube and to come in contact with hatching mouse blastocysts at the other end of the tube. The blastocysts were then washed and analyzed for the presence of the foreign DNA fragments. SETTING: Clinical and academic research environment. MAIN OUTCOME MEASURE: Detection of amplified products from transferred foreign DNA using the polymerase chain reaction and primers targeted at the E6-E7 region for different HPV types. RESULTS: Polymerase chain reaction analyses showed transference of DNA HPV type 18 to the blastocysts. Not all types of DNA fragments were transferred equally. CONCLUSION: The results suggested the possibility of using sperm as a noninvasive gene delivery system for passing on gene fragments to preimplantation embryos. It was demonstrated that certain DNA fragments were easier to deliver than others, indicating the necessity for exploring all the factors involved in the mechanism of the transference process. The study also serves to highlight the possibility of unintentional transmission of viral or bacterial DNA to the developing embryo via the sperm.

A new concept for anastomosis of the fallopian tube: tissue reconstruction with nonpenetrating, arcuate, legged clips in the rat model.

OBJECTIVE: To evaluate the titanium Kirsch-Zhu microclip microsurgical reanastomoses of the fallopian tubes. DESIGN: Compare the reanastomoses of the rat uterine horn between Kirsch-Zhu clips (Cushman Engineering, Albuquerque, NM) (group A) and conventional suture microsurgical techniques (group B). SETTING: Microsurgery Research Laboratory, Loma Linda University Medical Center, Loma Linda, California. PATIENTS, PARTICIPANTS: Fifteen SD rats Harlan (Harlan Sprague-Dawley Corp., Indianapolis, IN) were done in groups A and B and six in control group (C). MAIN OUTCOME MEASURE: Pregnancy rate, litter size, tissue of procedure, and histologic results. RESULTS: The clip technique was shorter in procedure time and resulted in equal fertility rate and litter size. Histologically, there were less granulomatous formation and histiocytic inflammation, but muscularis thinning and fibrosis were noted with the clip. CONCLUSIONS: The Kirsch-Zhu clip has potential for application to human fallopian tube reanastomoses.

Nonpenetrating, arcuate-legged clip reconstruction of the rat uterine horn.

A new method for reconstructing rat uterine horn was developed in which nonpenetrating, arcuate-legged clips are applied in interrupted fashion to everted seromuscular edges, forming an elastomeric flanged joint. This anastomosis has unusual physical and morphologic properties, with improved tissue healing and luminal restitution. Clipping is easier than suturing, and resulted in equivalent fertility rate (50-60%) and litter size. It also is associated with less granuloma formation and hystiocytic infiltration than suture. The new technique has the potential of endoscopic translation for human tubal reconstruction.

Human papillomavirus gene sequences in washed human sperm deoxyribonucleic acid.

The present study demonstrated the presence of HPV gene sequences in Percoll-washed sperm cells using polymerase chain reaction primers targeting smaller gene regions. Up to 64% of the sperm specimens were shown to contain gene sequences indicative of the presence of HPV. Human papillomavirus type 16 was detected about twice as often as HPV type 18. The results suggest the possible role of sperm as a vector for HPV.

Uptake of exogenous human papilloma virus L1 DNA by oocytes and detection by the polymerase chain reaction.

OBJECTIVE: The purpose of this study was to determine if oocytes were capable of taking up exogenous DNA such as human papillomaviral (HPV) DNA and evaluate the zona pellucida as a barrier to the entry of foreign DNA into the oocyte. METHODS: The experiment consisted of four groups of hamster oocytes exposed to HPV DNA fragments: Group A, zona-free oocytes (n = 5); Group B, oocytes with an intact zona pellucida (n = 5); Group C, oocytes fixed in 4% buffered formalin solution for 20 min (n = 5); and Group D, zona-free oocytes (n = 4). Group C oocytes served as an internal control to ensure adequate washing of the oocytes after incubation. RESULTS: The zona pellucida was not a barrier to foreign DNA molecules. The PCR did not detect L1-HPV and beta-globin gene sequences in the untreated hamster oocyte. Uptake of the smaller DNA fragments such as that amplified from the beta-globin region was independent of active oocyte cell processes. CONCLUSION: Oocytes cultured in vitro can passively take up exogenous DNA fragments. The results suggest a possible role of oocytes as vectors for foreign DNA.

Association of human sperm nuclear decondensation and in vitro penetration ability.

Sperm nuclear decondensation is an integral step in fertilization which leads to the formation of the male pronucleus. The association between the in vitro spontaneous nuclear decondensation of human sperm and its fertilizing ability was studied in infertile male patients. The ability of sperm to fertilize an egg using the discontinuous two-layer Percoll method was significantly correlated to the percentage of decondensed swollen head (r = 0.43; P less than 0.005). The fertilizing ability of sperm processed with Test-Yolk buffer was correlated with the percentage of sperm at the fully decondensed stalk stage (r = 0.51; P less than 0.05). There were insignificant correlations for the whole-wash centrifugation, cryopreserved-thawed and swim-up methods. Samples of sperm that were positive (greater than 0% fertilization) in the sperm penetration assay had a higher percentage of decondensed sperm heads (66.7% vs. 20.6%) after Percoll wash or whole-wash centrifugation (60.5% vs. 44.3%) treatments compared with samples with no fertilization. Treatments that included Test-Yolk resulted in high percentages of decondensed swollen heads. The results suggest a positive association between sperm nuclear decondensation and the fertilizing ability of sperm, and affirm the importance of nuclear decondensation to the study of fertilization events.

Follicular phase hormonal profiles during administration of leuprolide for in vitro fertilization.

Hormonal changes induced during short-term administration of leuprolide were evaluated during the follicular phase in 57 patients who completed an IVF cycle. They were compared with those of 14 patients who were placed on long-term suppression. There was an unexpected abnormal increase in serum progesterone during the first week of the cycle in nine of the 57, with no significant change in the fertilization and cleavage rate; however, no pregnancy was achieved in this group. Transient mild elevation of progesterone was also detected in 16 patients with no adverse effect on fertilization and the outcome of the IVF. In the long protocol, the tonic levels of LH, FSH, and progesterone remained low throughout the follicular phase. The total number of pregnancies was higher in the short suppression regimen, but the full-term pregnancy rates were similar in both protocols.

Diadenosine tetraphosphate (Ap4A) and triphosphate (Ap3A) signaling of human sperm motility.

The ubiquitous dinucleotide polyphosphate, diadenosine tetraphosphate (Ap4A), has been shown to be a signal molecule for DNA replication in mammalian cells. In this study, Ap4A and a related compound, diadenosine triphosphate (Ap3A), were tested for possible signaling functions in human spermatozoa. A computerized automated semen analyzer was used to detect changes in spermatozoa motility parameters. Cryopreserved-thawed donor spermatozoa were washed and incubated in 0.1 mM Ap4A, 0.1 mM Ap3A, or control medium. The data indicated that both Ap4A and Ap3A decreased the percentage of motile spermatozoa after 4 or more hours of incubation in vitro. The two dinucleotide polyphosphates caused an increase in the amplitude of lateral spermatozoa head displacement parameter only at the start of incubation. The other spermatozoa kinematic parameters were unaffected. No opposing ying-yang dual actions of Ap4A to Ap3A were seen. From the results, Ap4A and Ap3A were observed to be potential inhibitory signals of spermatozoa motility after prolonged exposure.

Cyclic CMP (cytidine 3',5'-monophosphate) suppresses changes in human sperm amplitude of lateral head displacement and hyperactivation.

The dibutyryl analog of cCMP suppressed sperm amplitude of lateral head displacement and hyperactivation. Sperm motility was inhibited by dibutyryl cCMP with a shift toward less linear trajectory sperm movements. The results suggest a role of cCMP as an inhibitory signal on sperm motility patterns related to sperm capacitation.

Inhibition of implantation in vitro of frozen-thawed mouse embryos by the signal molecule diadenosine tetraphosphate.

The dinucleotide polyphosphate, diadenosine 5', 5'''-P(1), P(4)-tetraphosphate (Ap4A), has been identified in mammalian and non-mammalian cells as a signal molecule that initiates the process of DNA replication and cell division. The objective of this study was to determine the function of this messenger molecule in preimplantation mouse embryonic cells. Frozenthawed two-cell mouse embryos were incubated in the presence of 0, 0.1 and 1.0 mM Ap4A at 37 degrees C in moist 5% CO(2) in air mixture for 5 d. The developmental stages of the embryos in terms of hatching and implantation were evaluated. The data showed dose-dependent inhibition of blastocyst implantation; however, there were no differences observed in the number of embryos developing to the blastocyst stage. The results suggest that Ap4A neither promotes nor inhibits the development of early stage embryos except at the implantation stage, where it exerts inhibitory control.

Effectiveness of stimulated menstrual cycles and Percoll sperm preparation in intrauterine insemination.

To examine the usefulness of intrauterine insemination in women with various fertility factors, we retrospectively analyzed data from women treated during 1986 and 1987. Ninety-three patients underwent 1-11 cycles of single or double procedures, for a total of 423 inseminations in 263 treatment cycles. Twenty-six patients (28%) conceived, for a 10% total pregnancy rate per cycle, with 58% of the pregnancies resulting from double inseminations per cycle. An average of two treatment cycles was required to achieve pregnancy. Eight pregnancies (31%) occurred in spontaneous cycles, while 18 (69%) occurred in stimulated cycles. While clomiphene citrate therapy was useful in anovulatory patients, it was of no benefit in ovulatory patients being treated with intrauterine insemination. Human menopausal gonadotropin therapy was of benefit in both ovulatory and anovulatory patients when combined with intrauterine insemination. The live birth rate was higher (75%) in spontaneous cycles than in stimulated cycles (44%). Semen preparation was accomplished by sperm washing in 61% of the pregnancies and by Percoll preparation in 39%. The effectiveness of the discontinuous Percoll gradient for semen preparation for insemination was suggested by a pregnancy rate of 9% per cycle. While the mean sperm count in the pregnant group was 44 million, successful pregnancy was accomplished with a double insemination of 880,000 and 1.16 million rapidly progressive sperm in the first and second inseminate, respectively. The data confirm the important role of intrauterine insemination for the treatment of infertility.

Evaluation of the Pipelle curette for endometrial biopsy.

The Pipelle endometrial suction curette was evaluated, and its application and effectiveness were compared to those of the Novak curette for endometrial sampling during the midluteal phase. Fifty women underwent an endometrial biopsy with the Pipelle and Novak curettes. Ninety percent of the women preferred the biopsy with the Pipelle. Histologically, tissue obtained with the Pipelle was satisfactory and similar to that with the Novak. The Pipelle curette appears to be an excellent device for midluteal endometrial biopsy during an infertility evaluation.

Effectiveness of short pituitary suppression with gonadotropin-releasing hormone agonist leuprolide during induction of ovulation for in vitro fertilization.

A short suppression regimen with daily 0.5 mg leuprolide commencing the first day of in vitro fertilization (IVF) cycles was evaluated in 10 women who previously underwent similar IVF cycle without suppression. Induction of ovulation, oocyte retrieval, incubation, and embryo transfer were similar in all the cycles. Assessment included the amount of human menopausal gonadotropin (hMG) used, length of stimulation, serum estradiol and luteinizing hormone (LH) levels, number of oocytes retrieved and their quality, cleavage rate, and number of embryos. The results showed that when leuprolide was used, no endogenous LH surge was detected, and there was a significant increase in hMG injected, from 19.0 +/- 5.8 to 34.4 +/- 17 ampoules, and in estradiol levels, from 1276 +/- 470 to 2618 +/- 1084 pg/ml (mean +/- SD). In addition, there was an increase in the total oocytes retrieved from 54 to 94, their cleavage rate from 59 to 86%, and the number of embryos from 24 to 70 in the suppressed cycle. No deleterious effects were observed and there were two pregnancies in this group.

Hirsutism: implications, etiology, and management.

Hirsutism usually results from a subtle excess of androgens. As such, it is a clue to possible endocrine disturbance in addition to presenting cosmetic problems. We use the term hirsutism to mean male-pattern hirsutism--excessive growth of hair in areas where female subjects normally have considerably less than male subjects. An elevation of the plasma free (unbound) testosterone level is the single most consistent endocrinologic finding in hirsutism. The plasma free testosterone level is sometimes elevated when the total level of plasma testosterone is normal because testosterone-estradiol--binding globulin (TEBG) levels are often depressed in hirsute women. Frequent blood sampling is sometimes necessary to demonstrate subtle hyperandrogenic states since androgen levels in the blood are pulsatile and seemingly reflect episodic ovarian and adrenal secretion. The source of hyperandrogenemia can usually be determined from dexamethasone suppression testing. Those patients whose plasma free androgen levels do not suppress normally usually have functional ovarian hyperandrogenism (polycystic ovary syndrome variants). Very high plasma androgen levels or evidence of hypercortisolism, which is not normally suppressible by dexamethasone, should lead to the search for a tumor or Cushing's syndrome. Those patients in whom hyperandrogenemia is suppressed normally by dexamethasone have a form of the adrenogenital syndrome, a prolactinoma, obesity, or idiopathic hyperandrogenemia. In such patients, glucocorticoid therapy may reduce hirsutism and acne and normalize menses. The treatment of hirsutism resulting from functional ovarian hyperandrogenism is not as satisfactory; estrogen-progestin treatment is the most useful adjunct to cosmetic approaches to hirsutism in this country. However, other manifestations of polycystic ovary syndrome, such as infertility, may take precedence over hirsutism when an optimal therapeutic program is designed for many patients.

Pituitary response to LHRH in hypothyroid women.

This study was designed to characterize pituitary function in premenopausal women during hypothyroid and euthyroid periods. Six subjects with basal thyroid-stimulating hormone (TSH) levels above 10 microU/ml were studied. Estradiol (E2), prolactin (PRL), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) levels were measured by radioimmunoassay at 30, 15, and 0 minutes before infusion. Pituitary function was evaluated by rapid infusion of 10 micrograms of luteinizing hormone-releasing hormone (LHRH) every 2 hours for a total of 6 hours. Samples were then obtained for FSH and LH every 30 minutes for the duration of the 6-hour study. A significant elevation in basal gonadotropins was observed during the hypothyroid period regardless of basal E2 levels in all 6 subjects (P < .01). Basal PRL levels were not significahtly different during the 2 periods (P > .05). In all 6 patients pituitary sensitivity and reserve correlated significantly with basal E2 levels (P < .05), but were not altered by the hypothyroid condition. It is concluded that pituitary responsiveness to LHRH is not altered in hypothyroid women, but that chronic elevation of basal gonadotropins may in part explain the anovulation that so often accompanies this disorder.

Hypothalamic-pituitary responses to high concentrations of gonadal steroids in a three-month-old infant with a feminizing gonadal stromal tumor.

A 3-month-old female infant feminized by an ovarian stromal tumor is presented. Clinicopathologic aspects of such tumors occurring in infancy are discussed. In addition, the evaluation of hypothalamic-pituitary function preoperatively and postoperatively are presented. In the preoperative, high-steroid environment, both basal and stimulated secretion of follicle-stimulating hormone (FSH) was suppressed. Secretion of luteinizing hormone (LH) was normal. Following surgical castration, stimulated secretion of both gonadotropins clearly increased. These findings suggest an active but less sensitive negative feedback mechanism for FSH than for LH during infancy, i.e., high concentrations of gonadal steroids are necessary to maximally suppress FSH secretion while normal steroid concentrations appear to maximally suppress LH secretion. Developmental changes in hypothalamic-pituitary sensitivity to negative feedback controls are discussed in light of these findings.

A reassessment of the fractional postcoital test.

The present investigation was designed to reassess the fractional postcoital test (PCT). Specifically, the study addressed the question of whether the internal cervical mucus sperm count is significantly different from the sperm counts at other cervical levels. By means of standardized methodology, 25 normal postcoital tests were perfoemed on 22 couples. Cervical mucus from three different levels within the endocervical canal was examined for total count, motility, and sperm morphology. No significant difference could be found in these three parameters among any of the cervical levels (p greater than 0.05). Significantly fewer abnormal sperm were found in the cervical mucus when compared to the total abnormal forms in the semen (p less than 0.001). Therefore, while the fractional PCT is a reflection of cervical sperm, this study indicates that the three different cervical levels in the fractional PCT (internal-mid-external) are equally effective in the interpretation of the postcoital test.

Unexplained infertility. A reappraisal.

During a 30-month period 229 couples were evaluated for infertility at the Naval Regional Medical Center, Oakland. Laparoscopy was used in all cases of otherwise unexplained infertility. The resulting laparoscopic examination of the female partner of 24 such couples demonstrated abnormal findings in 18 (75%). Of these 18 subjects, unsuspected endometriosis was found in 11 (46%) and peritubal adhesions in 7 (29%). Of the 229 couples evaluated, only 8 (3.5%) failed to show some etiologic factor associated with infertility. Thus, the previously reported 10 to 20% incidence of unexplained infertility is too high an estimate in view of the additional information made available through pelvic endoscopy.