Endometrial fluid collection in women with PCOS undergoing ovarian stimulation for IVF. A report of four cases.

BACKGROUND: The presence of endometrial fluid collections' developing during ovarian-stimulation was previously reported to occur in women with hydrosalpinx. We report on the occurrence of endometrial fluid collections in four women with polycystic ovary syndrome (PCOS) undergoing ovarian stimulation for in vitro fertilization. CASES: Four women developed endometrial fluid collections during ovarian stimulation. These fluid collections were noted as early as day 5 of stimulation. The reproductive outcome when fluid collections were noted on the day of human chorionic gonadotropin (hCG) or of embryo transfer (ET) was poor. One of three women with fluid collection on the day of hCG conceived but had a missed abortion. One patient with fluid on the day of ET failed to conceive. Three of four patients who underwent repeat cycles conceived when no fluid collections were seen on day of hCG or ET. CONCLUSION: Abnormal endometrial milieu could be an underlying defect in some women with PCOS and chronic anovulation who fail to conceive with ovulation-induction agents. This is the first report of endometrial fluid collections in patients with PCOS in the absence of hydrosalpinx. Continuous monitoring of the endometrial lining during ovulation induction is mandatory to rule out any abnormality in endometrial development. Cryopreserving all embryos may be considering in cycles with fluid collections noted on the day hCG or ET.

Effect of a serum extender containing growth factors on development of IVM and IVF bovine embryos.

The present experiments were conducted to determine if supplementation of the culture medium with a serum extender containing growth factors would increase development of bovine embryos into morulae or blastocysts, following in vitro maturation (IVM) and in vitro fertilization (IVF). In Experiment 1, bovine zygotes were cultured in CR1 medium supplemented with 0, 0.01, 0.1, 1 or 10% serum extender. In Experiment 2, bovine zygotes were cultured in the presence of cumulus cells in CR1 medium supplemented with 0, 0.01, 0.1, 1 or 10% serum extender. In Experiment 3, bovine oocytes were matured in Medium 199 supplemented with 0, 0.01, 0.1, 1 or 10% serum extender. In Experiment 4, oocytes were matured in Medium 199 with 10% fetal bovine serum (FBS) or 5% FBS with serum extender. Following maturation, zygotes were cultured in CR1 medium with 10% FBS or 5 % FBS and serum extender. In all 4 experiments, the embryos were cultured in vitro until Day 7 after IVF, and development to the morula or blastocyst stage was assessed. The findings of the first 2 experiments showed that the serum extender did not directly influence embryo development but did stimulate development when cumulus cells were included in the culture system. The remaining 2 experiments showed that the serum extender did influence development through its interactions with cumulus cells during maturation and/or culture. These findings suggest that although growth factors or other products do not directly stimulate bovine embryo development their effects may be mediated through secondary cell systems.

Established cell lines and their conditioned media support bovine embryo development during in-vitro culture.

These experiments were conducted to evaluate the ability of different somatic-cell monolayers or conditioned medium from somatic cells for supporting bovine embryo development in vitro. In the first experiment, bovine embryos (2- to 4-cells) were allocated randomly to a control (medium 199 with 10% fetal bovine serum and antibiotics) group or co-cultured with bovine oviduct epithelial (BOEC), buffalo rat liver (BRL), Madin Darby bovine kidney (MDBK) or African green monkey kidney (Vero) cells. In the second experiment, bovine embryos (1-cell) were allocated randomly to the following groups: control medium or conditioned medium from BOEC, BRL, MDBK and Vero monolayers. In both experiments, development to the blastocyst stage was assessed after 8 days of incubation at 39 degrees C and 5% CO2. In Experiment 1, coculture improved development to the blastocyst stage compared with control medium alone, and the highest development was observed after co-culture with BOEC. In Experiment 2, conditioned medium enhanced development to morulae and blastocysts compared with the control medium; however, no differences were detected among different cell supports. These results indicate that both co-culture and conditioned medium from different cell monolayers supported development to the blastocyst stage at a higher efficiency than control medium alone.

Frozen-thawed cumulus-granulosa cells support bovine embryo development during coculture.

OBJECTIVE: To evaluate the ability of bovine cumulus-granulosa cells to survive cryopreservation and subsequently support bovine embryo development during coculture. DESIGN: In vitro-matured and -fertilized bovine embryos (two- to four-cell) were allotted randomly to one of three treatment groups: [1] control medium alone consisting of Medium 199 containing 10% fetal bovine serum and antibiotics, [2] cocultured on fresh bovine cumulus-granulosa cells in control medium, or [3] cocultured on frozen-thawed cumulus-granulosa cells in control medium. Embryo development was assessed on days 7 and 8 after IVF. RESULTS: Coculture improved embryo development on days 7 and 8 compared with the control group. However, embryo development on days 7 and 8 did not differ among coculture groups. CONCLUSIONS: Frozen-thawed cumulus-granulosa cells enhance embryo development similar to fresh cells during in vitro coculture.

Predictive value of sperm hyperactivation measurements based on the dilution effect method in clinical in vitro fertilization.

Sperm hyperactivation motility characterized by wide oscillatory movements of the sperm head, nonlinear directions, and rapid motility with occasional star-shaped pattern of movement was measured during routine semen analyses prior to an in vitro fertilization procedure. The method consisted of diluting liquefied semen 1:20 with Ham's F-10 supplemented with processed human cord sera followed by incubation at 37 degrees C for 30 minutes. At the end of the incubation period, aliquots of semen samples were evaluated by phase contrast microscopy for sperm hyperactivation. The results indicated that (1) sperm samples exhibiting 15% or more hyperactive motility were associated with a significantly higher percent fertilization of oocytes during the IVF procedure; (2) sperm hyperactive motility was correlated to sperm fertilizability during IVF treatment cycles.

Hysterosalpingography, laparoscopy and hysteroscopy in infertility. A comparative study.

The accuracy and significance of hysterosalpingography (HSG) during an infertility evaluation were assessed by comparing the radiologic findings on HSG to the operative findings during laparoscopy and hysteroscopy. One hundred ninety-three patients underwent a complete infertility evaluation at our center. HSG was performed during the proliferative phase and was followed by laparoscopy and hysteroscopy, when indicated, during the same or next cycle. False-positive findings on HSG were noted in 5.1% of the patients. In 21%, adnexal adhesions and pelvic endometriosis were identified during surgery in spite of normal HSG. HSG is as accurate as laparoscopy in the diagnosis of tubal disease. However, laparoscopy excels HSG in the diagnosis of pelvic pathology. HSG should remain an integral part of the female infertility investigation and must be performed before laparoscopy and hysteroscopy.

Peritoneal fluid from patients with unexplained infertility inhibits growth of 2-cell mouse embryos.

A significantly higher DHEAS concentration was measured in the peritoneal fluid of unexplained infertility patients (1171.4 +/- 155 ng/mL) in comparison to normal controls (667.6 +/- 82 ng/mL). Since the androgenic male serum does not promote blastocyst formation in the mouse embryo assay system, the potential of growth impairment by peritoneal fluid (PF) obtained from 22 women with unexplained infertility and 10 fertile controls was assessed. Where peritoneal fluid and serum from unexplained infertile (UI) patients were used as media supplement in mouse embryo culture, a significant inhibition of growth was observed in dishes containing PF but not serum. When DHEAS was added in varying concentrations to the culture media, a dose-dependent inhibition of embryo growth was observed. These findings show that the elevated DHEAS concentrations in the PF of UI patients adversely effect embryo growth and further suggest that increased DHEAS levels in the cul-de-sac fluid may be a causative factor for infertility.

Peritoneal fluid fractions from patients with endometriosis do not promote two-cell mouse embryo growth.

Peritoneal fluid (PF) from 10 infertile patients with endometriosis, obtained during the follicular phase of the cycle during laparoscopy, did not promote two-cell mouse embryo growth to the extent observed by fluid obtained from seven normal controls. Five molecular weight (MW) fractions were obtained by ultrafiltration, and each was used as media supplement in the assay and compared with PF fractions from normal controls. All fractions of PF from patients with endometriosis inhibited mouse embryo growth to a greater extent than did normal controls. However, the MW fractions greater than 100,000 daltons showed greater inhibition of embryo development than did fractions less than 100,000 daltons. This study of cell-free PF suggests the presence of a humoral factor greater than 100,000 daltons that is inhibitory on mouse embryo growth.

Luteinizing hormone bioactivity and variable responses to clomiphene citrate in chronic anovulation.

In 18 women with infertility and chronic anovulation with normal gonadotropins, three different responses were observed to increasing doses (250 to 750 mg) of clomiphene citrate (CC). Follicle development and ovulation in 8, follicle development but no ovulation without human chorionic gonadotropin (hCG) in 6, and no response to CC in 4. Serum concentrations of bioactive luteinizing hormone (bioactive-LH), immunoactive (immunoactive-LH), follicle-stimulating hormone, and estradiol (E2) were measured and follicle growth was assessed by daily ultrasound. Findings were compared with 8 normal ovulatory controls. Folliculogenesis on CC therapy, based on our data, was 78%; however, only 44% ovulated spontaneously, 34% required hCG for follicle rupture. There were no apparent hormonal indicators to predict responders from nonresponders. The absence of an LH surge in the presence of follicles and sustained high E2 concentrations in 34% of patients may be associated with a decreased E2 sensitivity at the hypothalamic-pituitary level. Ultrasound easily identified patients who responded to CC with folliculogenesis but did not initiate an LH surge. Follicle rupture was achieved promptly by hCG administration.

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.

Pain relief in hysterosalpingography. A comparison of analgesics.

Hysterosalpingography provides important information in the evaluation of infertility but is generally considered an uncomfortable and painful procedure. We evaluated various analgesics for decreasing or eliminating the discomfort from this procedure. Two types of analgesia were required to give maximum pain relief during and after the examination in the 180 patients evaluated. The best results were achieved with a combination of naproxen sodium, 550 mg, given orally two hours before the examination, and 20% benzocaine, applied to the cervix.

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 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.

Continuous estrogen/progestin therapy in menopause.

The incidence of vaginal bleeding, endometrial histology, menopausal symptoms, blood pressure, and serum lipid concentrations were evaluated in 26 women who received either continuous conjugated equine estrogens and medroxyprogesterone acetate (group I, N = 16) or cyclic conjugated equine estrogen + medroxyprogesterone acetate (group II, N = 10) over a 9-month treatment period. At the end of therapy, endometrial biopsy specimens in group I revealed inactive endometrium, whereas one of ten biopsy specimens in group II showed proliferative endometrium. Blood pressure, serum total cholesterol, high-density and low-density lipoprotein cholesterol, and triglyceride levels were not significantly altered from baseline in either group during the 9-month treatment. Vaginal bleeding was virtually eliminated in group I by the end of the study, while 80% of the patients in group II continued to have cyclic menses (p less than 0.001). This pilot study suggests that continuous conjugated equine estrogen plus medroxyprogesterone acetate therapy appears to be a satisfactory method of postmenopausal hormone replacement, effectively reducing menopausal symptoms without any apparent short-term alterations in serum lipids. It has the added benefit of eliminating cyclic menstrual flow.