Search

Objective: To evaluate retrospectively the use of serum FSH levels and to correlate them with follicular growth in a clinical ovulation induction program., Methods: Twenty women with infertility due to anovulation associated with polycystic ovary syndrome (PCOS) were studied. The patients were down-regulated with a long GnRH agonist protocol and stimulated with purified urofollitropin, using a low-dose step-up regimen. Repeated serum samples were drawn and transvaginal ultrasound scans were-performed. During the exogenous FSH therapy serum FSH levels resulting in continuous follicular growth were analyzed, as well as the rates of ovulation, pregnancy, cancellation and conversion to in vitro fertilization (JVF)., Results: Thirty-two out of fifty treatment cycles led to ovulation, resulting in five term pregnancies. Eight cycles were converted to IVF/embryo transfer due to multiple follicular growth. They resulted in two pregnancies. Ten cycles were cancelled because of impaired follicular growth. The serum FSH levels (median 6 IU/I) resulting in continuous growth of the follicles were relatively stable within patients (variation 15%) but varied considerably between patients (45%). The relationship between FSH dose and serum level was different for lean and obese PCOS patients after subcutaneously injected urofollitropin, Conclusions: There seems to be a difference in resorption/metabolism between lean and obese PCOS patients with regard to s.c. injected FSH. The intra-patient coefficient of variation (C.V.) of the serum FSH response level was quite low, as was the C.V. of the FSH dose at the response level. This allowed a more rapid dose adjustment in subsequent cycles. Analysis of serum FSH during induction of ovulation with gonadotropins seems to be of limited value in clinical programs.

The objective of the present longitudinal descriptive study was to extend previous observations on the benefit of in-vitro fertilization (IVF) in cases of anejaculatory infertility due to spinal cord injuries (SCI) and to report results achieved by intracytoplasmic sperm injection (ICSI). The study was performed in a national referral unit for SCI, Spinalis SCI Research Unit, the Karolinska Institute. The patient material consisted of couples with SCI men seeking treatment for their infertility. The inclusion criteria were: stable relationship, motile spermatozoa in a diagnostic sample and no female contraindications. Spermatozoa were retrieved through electroejaculation or vibratory stimulation. If the sperm quality was judged to be sufficient, standard IVF was performed. ICSI was employed if the semen quality was extremely poor. We have treated 25 couples in 52 cycles, leading to 81 ovum retrievals and 47 embryo transfers. Total sperm counts were very variable (0.01-978 x 10(6)). Before the introduction of ICSI the fertilization rate was 30%. ICSI increased the fertilization rate to 88%. There was no association between the pregnancy rate and the sperm count, level of injury or fertilization technique. A total of 16 clinical pregnancies was established, leading to 11 deliveries. This gives a cumulative pregnancy rate per couple of 56%.

Background: The possibility of treating male infertility because of obstructive azoospermia has been poor, but intracytoplasmic sperm injection (ICSI) has given this type of infertility sufferer a new option., Material and Methods: In this study 13 couples with obstructive azoospermia were treated in a total of 19 stimulated IVF cycles. The men were between 27 and 45 (mean 33) years of age. Their partners, 24-39 (mean 31) years of age were treated according to routine IVF procedures, i.e. down regulation with buserelin followed by hyperstimulation with urofollitropin. Fertilization was obtained by ICSI. Two embryos were transferred on day two after the ovum pick up. Sperm were retrieved through microsurgical epididymal aspiration (MESA) in four cycles, percutaneous epididymal sperm aspiration (PESA) in three cycles and through testicular sperm extraction (TESE) in 12 cycles., Results: The overall fertilization rate was 68%, with a cleavage rate of 82%.The fertilization rate was equal (68%) with epididymal and testicular sperm and the cleavage rate was 87%) and 80%, respectively. Embryos were obtained for embryo transfer (ET) in all cases and five pregnancies (one twin pregnancy) were established (26% per ET), three using epididymal sperm and two using testicular sperm., Conclusion: Infertility due to obstructive azoospermia can successfully be treated with epididymal sperm and ICSI. When epididymal sperm cannot be found sperm extracted from a testicular biopsy can be used. PESA and TESE are quicker and easier alternatives to MESA and can be performed on an outpatient basis with local anesthesia.

We were interested in the degree of downregulation in women with poor response to in vitro fertilization (IVF) treatment. Ten women who were poor-responders, arbitrarily defined as having fewer than three follicles with diameter > 14 mm on days 9 and 10 of urofollitropin therapy, were compared with ten women who were not poor-responders, in a standard IVF program. The serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and estradiol after pituitary down-regulation did not differ between the two groups. The daily dose and the serum level of FSH were higher in poor-responders and there was a significant correlation between the dose of urofollitropin and the serum FSH level. There was no difference in serum LH level during ovulation stimulation between the groups. The clinical pregnancy rate was similar in the two groups although fewer embryos were transferred in the poor-response group. In conclusion, there was no difference with regard to pituitary suppression in normal-responders and poor-responders.

Forty normally ovulating women aged 25-38 years from one private and two university in vitro fertilization (IVF) centres were used in this randomized, double-blind, parallel, placebo-controlled study to explore the effect of recombinant human growth hormone (GH) on follicular fluid (FF) levels of steroid hormones, particularly androgens. All the women had tubal factor infertility and were classified as poor responders with at least two previously performed and failed IVF treatments in which less than five oocytes had been retrieved following ovarian hyperstimulation. Growth hormone (GH 0.1 IU/kg body wt per day) or placebo was given as pretreatment during down-regulation with gonadotropin-releasing hormone agonist and during stimulation with human menopausal gonadotropin (hMG) according to the randomized protocol. Follicular fluid concentrations of steroids were measured and changes related to the levels of insulin-like growth factor I (IGF-I) and IGF binding proteins 1 and 3 and to the mode of GH administration. Pretreatment with GH, i.e. administration of GH before hMG stimulation only, caused significantly elevated follicular fluid concentrations of estrone, testosterone and dehydroepiandrosterone (DHEA) and higher values for markers of aromatase activity (ratios between estrone and androstenedione and between estradiol-17 beta and androstenedione) than in the placebo group, as well as in the two groups receiving GH during hMG stimulation. The highest values for markers of steroid sulfatase activity (ratios between DHA and DHEA sulfate and between unconjugated and conjugated estrone) were found in the patients pretreated with GH. Positive correlations were found between follicular fluid IGF-I and IGF binding protein 3 on the one hand and androgens on the other. This study showed that the administration of adjuvant GH to women who were poor responders to gonadotropins alters the endocrine/paracrine ovarian response to gonadotropins.

Background: Assisted reproduction implies increased risks of pathological pregnancy, necessitating close follow up of early pregnancy. The use of serum hCG levels two and three weeks after embryo transfer for prediction of pregnancy outcome after in vitro fertilization-embryo transfer (IVF/ET) or gamete intrafallopian transfer (GIFT) was evaluated., Methods: Three hundred and twenty-nine treatment cycles were included. Serum samples were obtained on days 14 and 21 after ET and hCG was determined by fluoroimmunoassay. Receiver operating characteristic (ROC) curves were analyzed to find cut-off levels of hCG giving maximal sensitivity and specificity, identifying a low risk group and a high risk group with regard to pathological pregnancy., Results: The group of patients carrying a viable pregnancy had significantly higher hCG levels two and three weeks after ET than the group of patients carrying a pathological pregnancy. Furthermore, the daily increase in hCG was higher. In multiple gestations, the levels of hCG were significantly higher compared to singleton pregnancies. Ninety per cent of the patients with an hCG level > or = 150 IU/L 13-15 days after ET carried a pregnancy to term (the low risk group). Conversely, 50% of the patients with hCG < 150 IU/L carried a pathological pregnancy (the high risk group)., Conclusion: A single determination of the hCG level two weeks after ET, combined with transvaginal ultrasound two to three weeks later, is a reliable follow-up. In cases of subnormal hCG levels (< 150 IU/L), a second hCG determination one week later followed by sonography is recommended.

In order to study the effects of recombinant human luteinizing hormone (LH) and recombinant follicle stimulating hormone (FSH) on the pre-ovulatory follicle, immature pregnant mare's serum gonadotrophin-treated rats were used. Follicles were isolated by dissection and incubated for 4 h in medium 199 containing different concentrations of the gonadotrophins. In control medium the follicle-enclosed oocytes remained arrested in prophase I, but in the presence of recombinant human LH or recombinant human FSH, a concentration-dependent increase in the proportion of oocytes which underwent germinal vesicle breakdown (GVB) was seen. The lowest effective concentration of recombinant human LH was 10-50 ng/ml, and at 1000 ng/ml 97% of the oocytes underwent GVB. Recombinant human FSH was also effective, albeit slightly less so than recombinant human LH. Follicular oestradiol accumulation was stimulated up to 5-fold, both gonadotrophins showing similar effectiveness. Follicular progesterone and testosterone accumulation was markedly stimulated by recombinant human LH, giving a maximal 20-fold and 15-fold increase respectively. A 5-fold stimulation of progesterone production was found with recombinant human FSH. When submaximal concentrations of recombinant human LH and recombinant human FSH were combined, an additive effect was found on oocyte meiosis and on oestradiol and progesterone accumulation. The results are in agreement with previous studies using pituitary gonadotrophin and show that both recombinant human LH and recombinant human FSH have intrinsic effects on oocyte meiosis and steroidogenesis. It is likely that in the physiological situation in vivo, both gonadotrophins contribute to regulate these events in the peri-ovulatory period.

Treatment of non-seminomatous testicular cancer often leads to infertility due to anejaculation/retrograde ejaculation and poor sperm quality. In these men spermatozoa may be obtained by transrectal electroejaculation (TE), but the optimal strategy for assisted procreation in these couples is not known. Our aim was to examine whether TE and conventional in-vitro fertilization (IVF) would be successful. A total of 10 couples, with long-standing infertility due to anejaculation or retrograde ejaculation after treatment for testicular cancer 5-14 years earlier, were referred to our unit. All men underwent diagnostic TE under general anaesthesia. Spermatozoa were recovered in nine cases. The antegrade fraction was prepared and used for IVF. Sperm quality was variable and conventional IVF was considered impossible in three cases. Altogether six IVF treatment cycles in six couples resulted in five pregnancies, of which four resulted in a delivery and one resulted in a spontaneous abortion. One additional pregnancy is ongoing after transfer of cryopreserved embryos. The fertilization rate was 54% (33/61) and the cleavage rate was 97% (32/33). No complications relating to the procedure have been encountered.

Infertility due to spinal cord injury (SCI) in males has been identified for decades as an area of major concern and techniques for assisted ejaculation are available. There has not been an overall consensus regarding which type of assisted procreation is the most appropriate for these couples. We describe here our experience from a programme based on assisted ejaculation combined with in vitro fertilization (IVF). Twelve couples have been treated so far and altogether 22 cycles with ovum pick-up have been completed. Fertilisation of the oocytes was obtained in 18 of these cycles. The overall oocyte fertilisation rate was 49%. Embryo transfer took place in 17 cycles, leading to seven clinical pregnancies. Four of the pregnancies are delivered or are ongoing, whereas three ended in first trimester spontaneous abortion. Thus our initial experience suggests that assisted ejaculation in combination with IVF is an effective option for these couples.

Objectives: To explore the effect of recombinant, human GH on follicular development and oocyte retrieval after gonadotropin stimulation with the addition of GH or placebo to a standard IVF treatment regimen. Further, to investigate whether GH is a more effective adjuvant if the standard treatment regimen is preceded by GH injections., Design: A randomized, double-blind, parallel, placebo-controlled study., Setting: The IVF unit at university hospital., Patients: Forty normally ovulating women, age 25 to 38 years, with infertility because of tubal factors and being classified as "poor responders" with at least two previously performed and failed IVF attempts., Interventions: Human, recombinant GH (Genotropin, Kabi Pharmacia, Uppsala, Sweden) or placebo (0.1 IU/kg body weight per day) was given SC as pretreatment during down regulation with GnRH and during stimulation with hMG according to the randomized protocol., Main Outcome Measures: Number of oocytes retrieved after stimulation, total amount of gonadotropin used, time required for stimulation, number of follicles developing, rate of fertilization, and cleavage in vitro. Further, the quality of embryos, development of the endometrium, rate of clinical pregnancy, and serum and follicular fluid (FF) concentrations of insulin-like growth factor I (IGF-I), insulin-like growth factor binding protein-1 (IGFBP-1), and IGFBP-3 were estimated., Results: The number of oocytes retrieved did not differ significantly between the groups, nor did the amount of hMG required for stimulation. The fertilization rate increased in patients who had received GH. Growth hormone caused a significant increase in serum and FF levels of IGF-I. An increase in serum IGFBP-3 could also be recorded in patients who had received GH., Conclusion: Although certain beneficial effects were noted in GH-treated patients, the overall results did not support GH as a clinically useful adjuvant treatment.

A twin pregnancy following in vitro fertilization-embryo transfer coincidentally at risk for the X-linked recessive Duchenne muscular dystrophy is described. First-trimester prenatal diagnosis by transabdominal chorionic villus samplings on the dichorionic placentae and molecular linkage analysis could exclude the disorder in both fetuses. Genetic counseling and prenatal diagnosis were particularly complex due to the twin pregnancy, the need for linkage analysis, and confined placental mosaicism 45,X/46XX in one of the fetuses. All parties should be aware that additional invasive diagnostic procedures in the second trimester might be required. It is proposed that, in similar situations, only one, arguably two, fertilized egg be transferred at a time to facilitate prenatal diagnosis and decision making for these rare couples. This problem, however, may be increasingly overcome by preimplantation diagnosis.

Recent studies have indicated that growth factors such as insulin-like growth factors (IGFs) increase the growth rate of cultured preimplantation embryos. We therefore hypothesized that the fallopian tube may produce IGFs which in turn participate in the regulation of preimplantation embryo development in vivo. In the present study we examined the expression of IGF-I in the fallopian tube. We demonstrated that IGF-I transcripts (7.0, 1.7, and 1.2-0.8 kilobases) were abundant in the fallopian tube. Immunoreactive IGF-I was most abundant in the epithelial cells in the fallopian tube, and IGF-I messenger RNA (mRNA) was detected in the luminal region of the fallopian tube. A solution hybridization assay was used to examine the regulation of IGF-I mRNA. The abundance of IGF-I transcripts changed markedly during the 4-day estrous cycle with the highest levels on the day of proestrus. The increase in IGF-I mRNA between the day of diestrus II and the day of proestrus was 4-fold (P < 0.01). The pattern of IGF-I mRNA expression in the fallopian tube resembled the pattern of ovarian estrogen production during the estrous cycle. The level of IGF-I mRNA decreased after hypophysectomy. The expression of IGF-I mRNA in the fallopian tube was dose-dependently regulated by estradiol, and a single sc injection of estradiol [5 micrograms/100 g body wt (BW)] increased the IGF-I mRNA in a time-dependent manner with a significant increase after 3 h (P < 0.01). The lowest estradiol dose tested (0.1 microgram/100 g BW) increased the expression after 6 h, whereas progesterone (5 micrograms/100 g BW) was ineffective. The presence of embryos in the fallopian tube did not statistically significantly influence the abundance of IGF-I transcripts as measured with a solution hybridization assay on RNA extracted from whole fallopian tubes. In order to determine possible targets for fallopian tube-derived IGF-I we examined the expression of IGF-I receptor mRNA. Northern blot analysis revealed that an 11-kilobase IGF-I receptor transcript was expressed in the fallopian tube. Using a reverse transcriptase-polymerase chain reaction, IGF-I receptor mRNA was also detected in the eight-cell but not two-cell preimplantation embryo. The present study demonstrates that IGF-I is produced in the fallopian tube and its expression is regulated by estradiol. Both the fallopian tube and the eight-cell preimplantation embryo express IGF-I receptors and are therefore potential target tissues.

Cyclic AMP (cAMP) is one of the key regulators of the resumption of oocyte meiosis (oocyte maturation) in several mammalian species. In this study we have examined the role for cAMP in regulation of spontaneous maturation of human oocytes. Oocytes were obtained in connection with gynaecological operations and cultured for 24 or 48 h. A significantly larger proportion (77%) of oocytes cultured in the presence of 1 mM dibuturyl-cAMP (dbcAMP) for 24 h were arrested in meiosis compared to oocytes cultured in control medium (25%). In addition, dbcAMP stimulated progesterone accumulation during 48 h of incubation and inhibited monolayer formation of the cumulus cells attached to the oocyte. The data presented in this study demonstrate that dbcAMP delayed the spontaneous maturation of human oocytes and stimulated progesterone production by the cumulus cells. These results indicate that cAMP may be important also for regulation of meiosis in human oocytes and confirm results obtained in other species.

During follicular maturation there is a co-ordinated hormonal regulation of the theca and granulosa cells. It is generally believed that granulosa cell proliferation and differentiation are promoted mainly by follicle stimulating hormone (FSH) and that luteinizing hormone (LH) regulates the function of the theca cells. The aim of the present study was to examine the effect of LH/human chorionic gonadotrophin (HCG) on steroid production in human thecal cells. Isolated follicles (5-20 mm) were obtained during the follicular phase of the menstrual cycle in 10 women undergoing gynaecological laparotomy for reasons unrelated to ovarian pathology. The leading follicle(s) was excised and dispersed cells of the theca interna layer were isolated through combined mechanical and enzymatic techniques. The thecal cells were cultured 4-6 days with and without LH/HCG. Medium levels of androstenedione, testosterone and progesterone were measured by radioimmunoassay. Isolated thecal cells, cultured for 6 days, showed a high sensitivity to stimulation by LH/HCG. Steroid secretion was highest during days 0-2 and then declined gradually. LH/HCG stimulated steroid production in a dose-dependent way with the maximal stimulatory effect of LH at a concentration of 1-10 ng/ml, and of HCG at 0.01-0.1 IU/ml. The important question, especially in clinical situations, of the optimal level of LH for normal follicular maturation, remains to be answered. The present study is compatible with the view that thecal cell steroidogenesis in vivo is close to maximally stimulated by normal basal LH levels.

Objectives: To investigate if human thecal cells contain messenger ribonucleic acid (RNA) encoding insulin-like growth factor I (IGF-I) and insulin receptors and if IGF-I and insulin could stimulate androgen production in thecal cells., Design: Poly-adenine+ RNA was extracted from fresh thecal tissue, and the expression of the genes encoding insulin and IGF-I receptors were analyzed. Isolated thecal cells were cultured 4 to 6 days with and without hormones., Setting: Procedures were performed in a university laboratory., Patients: Eight women in the follicular phase of natural cycles were undergoing gynecological laparotomy for reasons unrelated to ovarian pathology. The leading follicle(s) was excised, and dispersed cells of the theca interna layer were isolated through combined mechanical and enzymatic techniques., Interventions: Luteinizing hormone (LH), IGF-I, and insulin were added to the cell cultures., Main Outcome Measure: The expression of IGF-I receptor and insulin receptor transcripts were analyzed by Northern blot. Medium levels of androstenedione and testosterone were measured by radioimmunoassay., Results: In the separated thecal tissue both IGF-I receptor and insulin-receptor transcripts were detected. Insulin-like growth factor I and insulin potentiated LH-induced androgen secretion while having less pronounced effects on basal androgen production., Conclusion: The present study demonstrates that both insulin and IGF-I receptor genes are expressed and that insulin and IGF-I can stimulate steroid production in human thecal cells. The study provides further support for the hypothesis that IGF-I and insulin may be involved both in physiological regulation of ovarian function as well as in its pathophysiology.

The endometrial thickness and echogenic pattern were prospectively evaluated in 100 patients undergoing IVF-treatment. On the day before oocyte aspiration (day-1) the endometrium was significantly thicker in the group of patients achieving an ongoing pregnancy than in the group that failed to conceive. The minimal endometrial thickness on day-1 compatible with an ongoing pregnancy was 9 mm. Before oocyte aspiration two different endometrial patterns could be distinguished, a multilayered, 'triple line', hypoechogenic endometrium (A) and an isoechogenic pattern (B). Other studies have, in addition, described a hyperechogenic endometrium with a poor pregnancy rate but that pattern could in the present study only be demonstrated after follicle aspiration, 32 clinical pregnancies occurred. Among patients with endometrial pattern A, 36% conceived and among patients with pattern B 23% conceived which was not significantly different. It is concluded that the day before aspiration an endometrial thickness of at least 9 mm appears to be required for an ongoing pregnancy. In all cases a triple line (hypo-or isoechogenic) pattern was evident before aspiration.

Both clinical and experimental evidence suggest that growth hormone may be of importance for ovarian function. The present study investigated whether growth hormone receptors are expressed in human granulosa cells. Granulosa cells were isolated either from natural cycles or from stimulated cycles in the course of in-vitro fertilization. Total RNA hybridized with a 32P-labelled rat growth hormone receptor cRNA probe revealed one major transcript with an estimated size of 4.5 kb and one minor transcript with an estimated size of 1.3 kb. Biotinylated growth hormone was used to analyse growth hormone binding. Competitive growth hormone binding was detected in freshly isolated granulosa cells, as well as in cultured cells. Growth hormone augmented basal and/or follicle stimulating hormone-stimulated steroidogenesis in granulosa cells obtained from patients with natural cycles, but the response to growth hormone stimulation showed considerable variation. We conclude that functional growth hormone receptors are present in human granulosa cells and that growth hormone, therefore, may have an important role in ovarian function.

In order to study the effect of insulin-like growth factor (IGF)-I on amino acid transport, granulosa-luteal cells were obtained from stimulated cycles in women undergoing in-vitro fertilization. The cells were precultured for 1-3 days in serum-containing medium. The medium was then changed to serum-free with added IGF-I and/or human chorionic gonadotrophin (HCG). Following 24 h culture, the cellular uptake of [14C]alpha-aminoisobutyric acid AIB (0.1 mM, 0.6 mCi/ml) was studied during a 2-6 h incubation. The results showed that IGF-I (10-100 ng/ml) consistently stimulated AIB uptake to levels which were 44 +/- 7% above the control (n = 11 experiments). The stimulatory effect of IGF-I was abolished by HCG and was reduced by IGF-binding protein. In conjunction with previous findings that IGF-I stimulates the uptake and incorporation of thymidine into DNA, these results suggest that IGF-I is involved in growth of human granulosa-luteal cells.

This study examines the possible direct effect of interleukin-1 alpha (IL-1 alpha) upon human granulosa cells. The cells were isolated from follicles of stimulated cycles in women undergoing oocyte retrieval for in-vitro fertilization. Purified cell preparations were cultured for different time periods in the presence of IL-1 alpha and human luteinizing hormone (LH) or follicle stimulating hormone. IL-1 alpha stimulated basal as well as LH-induced progesterone accumulation. The response in terms of cyclic AMP was more complex, there was no effect of IL-1 alpha on basal cyclic AMP accumulation. However, at the highest concentration tested (50 IU/ml), IL-1 alpha enhanced cyclic AMP accumulation over that seen with LH alone. At a lower concentration, IL-1 alpha either had no effect or was slightly inhibitory to the LH-induced cyclic AMP accumulation, depending on the culture period. Our results, taken together with other findings, are compatible with the view that IL-1 alpha has a potential regulatory role in the granulosa-luteal cell transition in the human ovary.

The effect of IGF-I on steroidogenesis in human granulosa cells was studied. Granulosa cells were obtained from follicles of both natural and stimulated cycles. The cells were cultured 4 to 6 days and the effect of IGF-I (1 to 100 micrograms/l) on basal, LH- and FSH-stimulated steroidogenesis was studied. It was found that in granulosa cells from follicles of natural cycles, FSH as well as IGF-I significantly stimulated progesterone and estradiol production in a majority of the experiments. A synergistic effect of FSH and IGF-I could be seen when low (1 and 10 micrograms/l) concentrations of the two hormones were used. Also in granulosa luteal cells from stimulated cycles a stimulatory effect of IGF-I on estradiol as well as progesterone production was observed. The present results suggest that IGF-I in combination with gonadotropins has a physiological role in the human follicle in controlling differentiation of the granulosa cells.

Human granulosa cells, obtained either from natural or stimulated cycles, were cultured, and the response to IGF-I and GH was analyzed. It was found that IGF-I alone stimulated thymidine incorporation in both types of granulosa cells. Furthermore, IGF-I alone and in combination with FSH or LH enhanced estradiol and progesterone production. In a limited series of experiments, GH in combination with FSH was found to stimulate steroidogenesis in granulosa cells obtained from natural, but not from stimulated, cycles.

Insulin-like growth factor I (IGF-I) has been proposed to be an autocrine/paracrine factor involved in granulosa cell proliferation and differentiation. The present study focuses on a possible mitogenic effect of IGF-I in human granulosa cells. Insulin-like growth factor I (1 to 10 ng/mL) significantly stimulated 3H-thymidine incorporation in granulosa cells obtained from both natural cycles and from patients stimulated for in vitro fertilization, whereas luteinizing hormone (LH, 10 ng/mL), follicle-stimulating hormone (FSH, 10 ng/mL) and epidermal growth factor (EGF, 10 ng/mL) had no apparent effect on deoxyribonucleic acid (DNA) synthesis under these conditions. Luteinizing hormone and FSH stimulated progesterone secretion whereas IGF-I and EGF were without effect. Our observations that IGF-I stimulates DNA synthesis in human granulosa cells is in agreement with previous reports, in other species, indicating that IGF-I might be of importance for granulosa cell proliferation.

The aim of the present study was to measure the level of cyclic GMP (cGMP) compared with the level of cyclic AMP (cAMP) in rat oocytes during resumption of meiosis I (oocyte maturation) and to microinject these cyclic nucleotides into the oocyte to study their effects on oocyte maturation. Immature oocytes were obtained from prepubertal rats primed with pregnant mare's serum gonadotropin. OOcytes were isolated adn short-term cultured under conditions enabling spontaneous maturation. The levels of cGMP and cAMP decreased in the oocyte during spontaneous maturation (from 0.41 to 0.25 and from 0.64 to 0.42 fmol per oocyte respectively). The decrease was observed during the first hour of culture, and no further decline was seen after 2 h. Microinjection of cGMP or cAMP into isolated immature oocytes delayed the spontaneous maturation, cGMP being slightly more effective than cAMP, whereas 2-deoxy-cAMP (which does not stimulate protein kinase A) did not. These results demonstrate for the first time that the level of cGMP decreases in the oocyte parallel to spontaneous meiosis, as already shown for cAMP. This suggests that cGMP, as well as cAMP, may be of importance for regulating this process. This assumption is further supported by data demonstrating a delay in the maturation of oocytes injected with cGMP.

Human granulosa cells from follicles of natural cycles (13 women) in mid- or late follicular phase were cultured in modified Medium-199. Human luteinizing hormone (100 micrograms/l), follicle-stimulating hormone (100 micrograms/l) and gonadotropin-releasing hormone agonist (GnRHa) (10(-12) to 10(-6) mol/l) alone or in combination were added to the culture medium. Medium content of progesterone was analysed. In granulosa cells obtained in the mid-follicular phase, the basal progesterone formation averaged 0.5 pg.cell-1.(48 h)-1. FSH caused a 3-fold stimulation. GnRHa (10(-6) mol/l) had a variable influence on the basal progesterone formation, whereas it consistently inhibited (40-50%) the FSH response. In granulosa cells from late follicular phase basal progesterone formation averaged 5 pg.cell-1.(48 h)-1 and was stimulated 3- to 6-fold by LH. GnRHa (10(-6) mol/l) stimulated the basal progesterone formation and caused a tended to potentiate the LH response on progesterone formation. It is concluded that GnRHa at relatively high concentrations exerts direct effects on gonadotropin-stimulated progesterone formation of human granulosa cells and that these effects are different (inhibitory or stimulatory) dependent on the degree of follicular maturation, and/or type of gonadotropin used.

The secretion of progesterone, testosterone, and oestradiol by intact human oocyte-cumulus complexes in vitro was examined in incubations lasting 6-24 h. The complexes were aspirated from preovulatory follicles in 32 women who, due to tubal disease, were participating in an in vitro fertilization program. In 12 of the women follicular maturation was induced with clomiphene and human chorionic gonadotrophin (hCG), in 13 women with human menopausal gonadotrophin (hMG) and hCG and in 7 women with a combination of clomiphene-hMG plus hCG. The net secretion of steroids into the fertilization medium was studied before (0-6 h) and after (6-24 h) the addition of sperm, by RIA of aliquots removed at specific times. A high and sustained secretion of progesterone was found both before and after insemination. Testosterone secretion remained at a low and constant level while a net release of oestradiol was found mainly during the first hours of incubation. The release of steroids, particularly progesterone, varied according to the mode of hormonal stimulation in vivo and was highest in complexes from clomiphene-hMG-treated women, probably reflecting different maturity of the aspirated follicles. In a second series of experiments the dispersed cumulus cells were recovered after fertilization and cultured as monolayers for 2-4 days. The cells underwent spontaneous luteinization and secreted high amounts of progesterone. These results extend previous work in animals showing that also in the human the periovulatory cumulus cells are steroidogenically active. The results also suggest a functional difference in the cumulus cells related to the mode of ovulation induction.

Rabbits were injected with 100 i.u. hCG to induce oocyte maturation. Oocytes removed from the ovaries at various times and denuded showed increased oxygen consumption which began before the start of maturation.

Human granulosa cells were isolated from preovulatory follicles during cycles stimulated with HMG-HCG or clomiphene-HMG-HCG or from unstimulated cycles. The cells were cultured for 6-8 days in medium M199 containing fetal calf serum under 5% CO2 in air. Highly purified human prolactin and human chorionic gonadotrophin were added alone or in combination to the cultures, and the content of steroids in the medium was measured every second day, utilizing conventional RIA techniques. In the presence of HCG the formation of progesterone (P) increased 3-5-fold over the control level with maximal effect after 4 days. In cells derived from clomiphene-HMG-HCG stimulated cycles, prolactin per se did not influence basal P formation but reduced the stimulatory effect of HCG. This was only seen in granulosa cells from follicles greater than 20 mm in diameter. In experiments with Forskolin, an adenylate cyclase activator, P formation was stimulated and the stimulation was counteracted by the concomitant presence of prolactin, indicating that prolactin did not interfere with the LH-HCG receptor. In cells from smaller follicles, or in cells from follicles aspirated from the natural cycle prior to the endogenous LH peak, P formation was stimulated by HCG but the addition of prolactin did not reduce this stimulatory effect. The results are discussed in relation to earlier reports on prolactin effects in vitro both on laboratory animals and human material.

The influence of cyclic nucleotides on meiosis in isolated rat oocytes was studied. It is known that dbcAMP inhibits the spontaneous maturation of isolated rat oocytes. However, the effect of other cyclic nucleotide derivatives upon meiosis has been poorly investigated. The present study shows that 8-BrcAMP is comparatively ineffective in inhibiting oocyte meiosis but exerts a potent stimulatory effect on progesterone production in cumulus cells. Neither dbcGMP nor 8-BrcGMP were able to inhibit the spontaneous maturation of cumulus-enclosed oocytes. On the other hand, 8-BrcGMP dose-dependently inhibited the meiosis of denuded oocytes. This indicates that the cumulus cells may be of importance for a possible regulatory role of cGMP. These results show the differences in meiotic response to various cyclic nucleotide derivatives.

Graafian follicles were extirpated before the endogenous LH surge on the day prior to ovulation from PMS-injected immature rats. They were incubated in chemically defined medium in presence or absence of gonadotrophins for 4-10 h. After incubation the oocyte-cumulus complexes were recovered from the follicles and one group inspected by Nomarski interference contrast microscopy and one group was placed in saline containing hyaluronidase. In hormone-free medium both the oocyte and the cumulus cells remained morphologically unchanged: a dictyate oocyte surrounded by a compact mass of cumulus cells. Hyaluronidase did not detach cells from the cumulus structure. However, in presence of LH or FSH morphological changes developed in both cell types: the oocyte resumed meiosis as revealed by germinal vesicle breakdown and polar body formation; the cumulus structure became dispersed and embedded in a viscous matrix. The individual cumulus cells displayed pseudopodia-like processes of the cell surface. Treatment with hyaluronidase resulted in detachment of cells from the cumulus. The effects of LH described on the oocyte and cumulus cells were unimpaired in presence of cyanoketone or aminoglutethimide although these agents blocked the follicular relase of progesterone, estradiol and androstenedione as revealed by RIA. The results indicate that a close functional relationship exists between the two cell types of the cumulus oophorus, the oocyte and the cumulus granulosa cells. Both cell types are affected directly or indirectly by gonadotrophins and characteristical morphological changes develop synchronously in both cell types.

Steroid production depends on the cholesterol (CH) substrate supplied by circulating lipoproteins which are internalised in the cells by receptor-mediated mechanisms. Low density lipoproteins (LDL) stimulate progesterone production in vitro. However, studies on follicles indicate low levels of LDL in follicular fluid (FF1). In the present study FF1 was obtained by ultrasound-guided punctures just before ovulation from 17 women participating in an in vitro fertilization programme. Serum was obtained simultaneously. Follicular development was stimulated with hMG-HCG or clomiphene-hMG-hCG combinations. In another 8 women FF1 was collected in connection with surgery for sterilization. FF1 levels of CH, triglycerides (TG), phospholipids (PL), apolipoprotein A1 and B (apoA1; apoB), oestradiol and progesterone were assayed in both groups as were the corresponding serum levels in the stimulated patient group. The FF1 levels of apoA1, TG and PL were approximately half of the levels in HDL in normal serum in both groups. However, CH was slightly lower in the stimulated group. ApoB was not detectable in FF1. Oestradiol was similar in both groups while progesterone was much higher in the stimulated than in the non-stimulated cycles. FF1 levels of apoA1 correlated positively to CH and PL in both groups and to progesterone in the stimulated follicles, while the correlation was negative in the other group. The absence of apoB and the levels of CH, TG, PL and apoA11 indicate that high density lipoprotein (HDL), but not LDL is present in FF1.(ABSTRACT TRUNCATED AT 250 WORDS)

It is known that dibutyryl cyclic AMP (dbcAMP) and theophylline inhibit the spontaneous maturation of isolated mouse oocytes. The present study demonstrates that dbcAMP (0.01-1.0 mM) as well as cyclic AMP (cAMP, 10 mM) and a phosphodiesterase inhibitor (IBMX, 0.01-1.0 mM) prevent spontaneous maturation of isolated rat oocytes. As reported earlier an increase in oxygen consumption by the oocyte was found following maturation. When the oocytes were cultured in the presence of dbcAMP or cAMP no change in respiration occurred during culture. These results argue against the theory that cAMP acts as a direct mediator of the action of luteinizing hormone (LH) on oocyte maturation. Furthermore they suggest that changes in oocyte energy metabolism are closely related to the maturation process.

The clinical experience in an IVF/ER program of a newly developed commercial kit (Estradiol enzymatique; BioMerieux) to determine urinary estrogens enzymatically, is reported. The study included 157 cycles, and the main indication for treatment was infertility due to tubal factor. Follicular development was stimulated in most cases by a combination of clomiphene citrate, human menopausal gonadotropin and chorionic gonadotropin. In addition to the previously used method to monitor follicular growth, ultrasound, daily urinary estrogens (estradiol + estrone) levels were assayed. A correlation between the pattern of urinary estrogen and the clinical results was found. In cycles with continuously rising estrogen up to the time of hCG administration a higher rate of in vitro fertilization and clinical pregnancies were obtained as compared to cycles where urinary estrogen declined or levelled off. It is concluded that this enzymatic assay of urinary estrogens is a good alternative to increase the precision of follicle monitoring when radioimmunological assay of serum estradiol levels cannot be obtained on a daily routine basis.

The present review briefly summarizes the current view of the mechanisms whereby prostaglandins (PGs) may act to modulate ovarian function. Particular concern is devoted to the aspects of ovarian function which are under current investigation by the authors, namely follicular maturation and rupture, granulosa cell and oocyte maturation, and the formation and maintenance as well as the regression of the corpus luteum (CL). PGs are formed in the granulosa cells of the preovulatory follicle in response to gonadotropin action and are at their highest levels around the time of ovulation. PGs are believed to be obligatory for follicular rupture, but their mechanism of action is still unknown. In the process of oocyte maturation PGs can mimic the stimulatory effects induced by LH or hCG, but are probably not obligate mediators of these effects. The importance of PGs for CL formation and maintenance is so far unsettled, and PGs may be regarded more as modulators than as inducers for these processes. The most well-documented effect of PGs is the induction of CL regression. Evidence is presented that PGs may have this effect also in the human.

Granulosa cells were aspirated from human pre-ovulatory follicles following a combined clomiphene-gonadotrophin stimulation in an in-vitro fertilization (IVF) programme. The cells were cultured for 8 days in medium M199 containing 10% bovine fetal calf serum under 5% CO2 in air. Pure human FSH and human LH were added alone or in combination to the culture in various concentrations and the progesterone (P) and oestradiol-17 beta (E2) levels in the medium were measured every second day by a conventional RIA technique. In the presence of FSH or LH the formation of P increased 2- to 3-fold with the pronounced effect after 4 to 6 days in culture. Addition of testosterone (T) (3 X 10(-7) M) to the culture medium affected neither basal nor gonadotrophin stimulated P formation. In this system, only minute amounts of E2 were formed and neither FSH nor LH stimulated its formation. When the medium was fortified with T, basal E2 formation increased 50- to 100-fold. FSH further stimulated this conversion significantly after 6 and 8 days of culture, while LH had no significant influence.

Previous studies have shown that gonadotrophin-releasing hormone (GnRH) can induce resumption of meiosis in follicle-enclosed rat oocytes. In the present study a GnRH antagonistic analogue ([D-pGlul,D-Phe2,-D-Trp3,6]LRF) was found to effectively abolish the stimulatory effect of a GnRH agonist upon resumption of meiosis and lactate accumulation in isolated pre-ovulatory rat follicles but the have no effect on LH stimulation of these parameters. It is concluded that although LH and GnRH can evoke a similar response they act through separate receptor sites and that it is unlikely that GnRH mediates the effect of LH on meiosis or glycolysis.

The effect of danazol on progesterone formation was studied in cultured human granulosa cells obtained from 20 follicles in the mid- to late follicular phase of normally menstruating women. The cells were cultured for 2 to 6 days in the presence of danazol (0.01 to 5 mg/l) alone and in combination with one of several progesterone stimulatory agents. The medium was changed every other day and analysed for progesterone with radioimmunoassay. In all cases progesterone synthesis was markedly stimulated by human FSH (10 micrograms/l), human LH (10-100 micrograms/l), hCG (1000 IU/l), forskolin (1 mumol/l) or 8-Bromo-cAMP (1 mmol/l). In the presence of danazol (1 mg/l) the FSH-, LH- and hCG-stimulated progesterone synthesis was partially inhibited by 55 to 60%. The response to forskolin or 8-Bromo-cAMP was also inhibited by danazol although to a somewhat lower extent (35 to 50%). Basal progesterone synthesis was inconsistently inhibited in some cases. It was concluded that not only gonadotropin-stimulated steroid synthesis, as previously demonstrated for hCG, but also forskolin and 8-Bromo-cAMP-stimulated steroidogenesis is sensitive to danazol inhibition. This suggests that the effect of danazol in human granulosa cells is due, at least partly, to interference with steps distal to cAMP generation.

Cultured human granulosa cells are dependent upon gonadotrophins and exogenous cholesterol for maximal progesterone production. In previous studies low-density lipoproteins (LDLs) have been considered the preferred source of cholesterol, while high-density lipoproteins (HDLs) seemed rather to reduce progesterone synthesis in these cells. In this study, granulosa cells were harvested from 12 women and cultured in medium containing lipoprotein-deficient serum for 3 days. This was followed by 2 days of culture in medium supplied with LDL or HDL fractions, prepared by sequential ultracentrifugation and affinity chromatography. In the presence of LH, both lipoprotein fractions enhanced progesterone secretion in a concentration-dependent fashion while basal progesterone secretion was enhanced to a lesser extent. Progesterone secretion in response to lipoproteins did not differ between cells from pre-ovulatory follicles stimulated with gonadotrophins in vivo and less mature follicles obtained earlier in the menstrual cycle. We conclude that human granulosa cells in vitro can utilize cholesterol carried by HDL as well as LDL for progesterone synthesis.