Search

background: Near infrared (NIR) spectroscopy is a technology proposed to facilitate non-invasive screening for the most optimal human embryo for uterine transfer. It has been proposed that the NIR spectral profile of an embryo’s spent culture medium can be used to generate a viability score that correlates to implantation potential. As the initial proof of principle studies were all retrospective, our aim was to investigate whether NIR spectroscopy on spent embryo culture medium in an on-site, prospective setting could improve the ongoing single embryo transfer (SET) pregnancy rate after Day 2 and 5 transfers. methods: We conducted a single-centre, double-blinded, randomized controlled trial in which the NIR group was compared with a control group. The primary outcome was the clinical pregnancy rate after 6–7 weeks of gestation per randomized patient. In the control group embryo selection was based only on traditional morphological evaluation while in the treatment group NIR spectroscopy was added to the morphological evaluation. results: The study was terminated early as the analysis of the Data Safety Monitoring Board showed a very low conditional power of superiority for the primary outcome. Of the 752 patients calculated to be included in the study, 164 and 163 patients were randomized into the NIR and control groups, respectively. No significant difference in the ongoing pregnancy rate per randomized patient was found between the NIR and the control group, 34.8 versus 35.6%, (P ¼ 0.97). The proportional difference between the study groups mean was 20.8% (95% confidence interval –11.4 to 10.2). conclusions: This study shows that adding NIR spectroscopy, in its present form, to embryo morphology does not improve the chance of a viable pregnancy when performing SET. The NIR technology appears to need further development before it can be used as an objective marker of embryo viability. clinical trials identifier: ISRCTN23817363.

BACKGROUND: It has been claimed that the risks to the child resulting from vitrification as compared with the slow-freezing technique, may be higher owing to the high concentrations of potentially toxic cryoprotectants. We therefore retrospectively compared the obstetric and neonatal outcomes in a cohort of children born after transfer of vitrified blastocysts, fresh blastocysts and slow-frozen early cleavage stage embryos. METHODS: All children born after transfer of vitrified blastocysts (n = 106), fresh blastocysts (n = 207) and slow-frozen early cleavage stage embryos (n = 206) during the period January 2006 to May 2008 at Fertility Center Scandinavia were included. Data on obstetric and neonatal outcomes were obtained from medical records from the antenatal and delivery clinics. RESULTS: For singletons, there were no significant differences between the groups in gestational age, mortality or birth defects. After adjustment for parity and BMI, birthweight was significantly higher in singletons born after transfer of vitrified blastocysts as compared with after transfer of fresh blastocysts (median 3560 versus 3510 g, P = 0.0311). More singletons born after transfer of fresh blastocysts were small for gestational age compared with singletons born after transfer of vitrified blastocysts (12.1 versus 3.0%, P = 0.0085). A higher rate of major post-partum haemorrhage was observed in the vitrified blastocyst group as compared with the other two groups (25.0 versus 6.0 and 7.5%). CONCLUSIONS: No adverse neonatal outcomes were observed in children born after transfer of vitrified, as compared with fresh blastocysts or after transfer of slow-frozen early cleavage stage embryos.

background: In a randomized controlled study aiming to test the effectiveness of preimplantation genetic screening (PGS) in women of advanced maternal age, embryos diagnosed as chromosomally abnormal and those with no diagnosis were fixed for reanalysis. The aim of this study was to determine how well the chromosomal constitution of one biopsied blastomere reflects the status of the entire embryo. methods: One hundred and seventy-three embryos diagnosed as chromosomally abnormal, 22 with no PGS result and four degenerated embryos originally diagnosed as normal were fixed and reanalysed by fluorescence in situ hybridization. results: In total, 199 embryos were fixed, of which 166 were successfully reanalysed. One hundred and sixty embryos were found to be chromosomally abnormal; 48 of the reanalysed embryos with an initial diagnosis (149) had at least one cell with exactly the same chromosomal constitution shown in the first PGS analysis (34.2%). The reanalysis confirmed the initial overall chromosomally abnormal status of the embryo in 95.9% of the cases. Of all chromosomally abnormal embryos, 4.1% were diagnosed as false positive. The risk for false negative rate was at least 4.1%. conclusions: PGS seems to be a good method for selecting against chromosomally abnormal embryos but not for determining an embryo’s exact chromosomal constitution.

BACKGROUND: Advanced maternal age (AMA) is an important parameter that negatively influences the clinical pregnancy rate in IVF, in particular owing to the increased embryo aneuploidy rate. It has thus been suggested that only transferring euploid embryos in this patient group would improve the pregnancy rate. The purpose of this study was to test whether employing preimplantation genetic screening (PGS) in AMA patients would increase the clinical pregnancy rate. METHODS: We conducted a two-center, randomized controlled trial (RCT) to analyze the outcome of embryo transfers in AMA patients (� 38 years of age) after PGS using FISH analysis for chromosomes X, Y, 13, 16, 18, 21 and 22. The PGS group was compared with a control group. The primary outcome measure was clinical pregnancy rate after 6–7 weeks of gestation per randomized patient. RESULTS: The study was terminated early as an interim analysis showed a very low conditional power of superiority for the primary outcome. Of the 320 patients calculated to be included in the study, 56 and 53 patients were randomized into the PGS and control groups, respectively. The clinical pregnancy rate in the PGS group was 8.9% (95% CI, 2.9–19.6%) compared with 24.5% (95% CI, 13.8–38.3%) in the control group, giving a difference of 15.6% (95% CI, 1.8–29.4%, P 5 0.039). CONCLUSIONS: Although the study was terminated early, this RCT study provides evidence against the use of PGS for AMA patients when performing IVF. Trial registration number: ISRCTN38014610.

BACKGROUND A prospective randomized study was carried out in two centres to compare the number of oocytes retrieved after two different starting doses of recombinant human FSH (rhFSH) (Gonal-F) in women undergoing ovarian stimulation for IVF/intracytoplasmic sperm injection (ICSI) cycles using the multiple dose regimen of the gonadotrophin-releasing hormone (GnRH) antagonist cetrorelix (Cetrotide) to prevent induction of the premature LH surge. METHODS Sixty women were randomized to receive rhFSH 150 IU ('low'), and 60 women to receive rhFSH 225 IU ('high') as the starting dose for the first 5 days of stimulation. From stimulation day 6 and onwards, including the day of human chorionic gonadotrophin (HCG) administration, the women received 0.25 mg of cetrorelix as a daily dose. The primary endpoint was the number of oocytes retrieved. RESULTS The mean number (+/- SD) of oocytes was 9.1 +/- 4.4 and 11.0 +/- 4.6 in the 'low' and 'high' groups respectively (P = 0.024). The mean number of 75 IU ampoules of rhFSH was significantly lower in the 'low' group (23.0 +/- 6.3 versus 30.5 +/- 5.6, P < 0.0001). The ongoing pregnancy rate per started cycle and per embryo transfer were 25.9 and 28.8% versus 25.4 and 26.8% respectively in the 'low' and 'high' rhFSH groups (P = NS). CONCLUSIONS When using a starting dose of 225 IU rhFSH combined with the multiple dose of 0.25 mg cetrorelix from stimulation day 6, significantly more oocytes were obtained than with a starting dose of 150 IU rhFSH.

The study compares treatment outcome and costs of ovulation induction cycles and in vitro fertilization cycles in infertile women with clomiphene resistant polycystic ovary syndrome.Twenty-eight infertile women with clomiphene resistant polycystic ovary syndrome referred to a university clinic were prospectively randomized to ovulation induction or in vitro fertilization. Forty-one ovulation induction cycles and thirty in vitro fertilization cycles were performed. Mann-Whitney U-test was used for between group comparisons and frequencies were compared with Fisher's exact test.More pregnancies per completed cycle were noted in the in vitro fertilization group than in the ovulation induction group. Drug costs were not much higher in the in vitro fertilization group but treatment costs were higher due to the additional costs of ovum pick up and embryo transfer. The cost per pregnancy was about twice as high in the ovulation induction group as in the in vitro fertilization group. The cost per term pregnancy including delivery was 1.6 times higher in the ovulation induction group.For a group of obese women with clomiphene resistant polycystic ovary syndrome, in vitro fertilization seems a cost-effective treatment.

Open testicular biopsy is a classic method of investigation in men with azoospermia. Recently, percutaneous needle biopsy of the testis has been used in attempts to obtain material for histopathological diagnosis in such cases and to retrieve spermatozoa for intracytoplasmic sperm injection (ICSI). To determine whether a 19 gauge (G) and a 21G butterfly needle could be used for percutaneous aspiration of testicular tissue to determine the presence of mature spermatids and assess spermatogenesis, 10 patients (16 testes) and 12 patients (17 testes) underwent 19G or 21G needle biopsy respectively, immediately followed by open testicular biopsy, with both procedures under local anaesthesia. Biopsy with each needle size was compared with open biopsy. With the 19G needle, in the 14 cases where material was obtained there was full agreement with open biopsy regarding the presence or absence of mature spermatozoa, whereas with the 21G needle only nine of the 13 biopsies yielding material were predictive in this respect. Each needle size correlated poorly with open biopsy regarding evaluation of spermatogenesis. We conclude that percutaneous biopsy with a 19G butterfly needle is a quick and reliable method for demonstrating spermatozoa for ICSI. But for a detailed histopathological diagnosis, however, the needle biopsies gave poor results, whereas the material from the open testicular biopsies was assessable.

Treatment of testicular cancer (TC) may cause infertility due to reduced sperm quality with or without an ejaculation problem. In cases of anejaculation or retrograde ejaculation, spermatozoa can be obtained by transrectal electroejaculation (TE) or testicular sperm extraction (TESE) and used for in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). In this study, 15 out of 17 couples evaluated for infertility after TC, underwent a total of 21 treatment cycles, resulting in 18 embryo transfers. Spermatozoa were obtained by TE in 16 cycles, by masturbation in three cycles and by TESE in one. In one cycle no spermatozoa were found using TESE. Fertilization and cleavage was achieved by IVF in seven cycles and ICSI in 11 cycles; average fertilization rates of 57 and 55% respectively were observed. Twelve clinical pregnancies occurred, of which 11 have been delivered or are ongoing. The ongoing pregnancy rate was 57% per cycle. These results show that infertility after testicular cancer can be treated effectively with IVF and that ICSI even permits treatment of patients who have severe oligozoospermia.

Purpose: The aim of this retrospective study was to compare the outcome of in vitro fertilization and embryo transfer in women with endometriosis and a control group with tubal-factor infertility. Methods: Forty-eight patients with endometriosis underwent 65 cycles of in vitro fertilization and embryo transfer at Huddinge University Hospital. The matched control group with tubal-factor infertility consisted of 98 cycles in 98 patients. These groups were retrospectively analyzed regarding stimulation, fertilization, embryo development, implantation, and pregnancy outcome. Results: The fertilization rate was significantly lower in women with endometriosis, but the cleavage, implantation, and pregnancy rates did not differ. Conclusions: Our results show that women with endometriosis have a lower fertilization rate compared with women with tubal-factor infertility. However, once the oocyte is fertilized, it seems that the preembryo has a normal chance of implantation, leading to similar pregnancy rates.

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.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.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.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 diameter14 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.

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.

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 38years, 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 (0.1IU/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.

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.

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 (P0.01). 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 (P0.01). 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.

Percutaneous epididymal sperm aspiration (PESA) for retrieval of spermatozoa for intracytoplasmic sperm injection (ICSI) is a new simplified technique in the treatment of men with obstructive azoospermia. There has been a fear that the PESA procedure, being blind, could cause damage to the epididymal duct system and make it impossible to retrieve spermatozoa if a repeated procedure is required. We report here on repeated PESA procedures from the same unilateral epididymis. Twenty-seven men with obstructive azoospermia were investigated retrospectively regarding sufficiency of the number of motile spermatozoa for ICSI, fertilization rate (FR) and possibility of collecting spermatozoa for cryopreservation in repeated PESA procedures. Sufficient motile spermatozoa for ICSI were found in a similar proportion of men at the first two attempts: 91 and 89% respectively. Fertilization rate and the possibility of collecting spermatozoa for cryopreservation were also similar at the first two PESA procedures: 62 versus 67% and 33 versus 33% respectively. At the third procedure, motile spermatozoa for ICSI were retrieved in 86% (6/7), FR was 47% and spermatozoa were cryopreserved in one case. Two men underwent a fourth PESA. In both cases, a sufficient number of motile spermatozoa for ICSI was found and FR was 62%. This study shows that in men with obstructive azoospermia, PESA can be repeated on the same unilateral epididymis up to three times, with good opportunity of retrieving sufficient motile spermatozoa for ICSI.

BACKGROUND: A pre-ovarian block (POB) technique can be used for pain relief during oocyte retrieval in IVF. The local anaesthetic is deposited in the vaginal wall and between the vaginal wall and the peritoneal surface near the ovary using ultrasound guidance. The aim of this study was to test whether analgesia with POB resulted in improved pain relief compared to paracervical block (PCB). METHODS: A prospective, randomized, multicentre study of POB versus PCB (10 ml of 1% lidocaine each) with 183 patients randomized to POB (n = 96) or PCB (n = 87) was performed. Randomization (via a computer-generated list) was balanced for age, previously completed IVF cycles, degree of anxiety, estimated number of follicles, BMI, premedication and centre. Pain was measured using a visual analogue scale (VAS, 0–100 mm) and given as median values. The primary end-point of this study was overall VAS pain score for both sides during the oocyte retrieval procedure. RESULTS: Overall pain during the entire oocyte retrieval was 22 (POB) and 16 (PCB) (P = 0.42). No differences were found in degree of anxiety, premedication, dose of alfentanil, fertilization rate, number of good-quality embryos or clinical pregnancy rate. CONCLUSIONS: No differences were found in overall pain experienced during the entire oocyte retrieval procedure with POB compared to PCB.

We have tested and subsequently successfully applied a single-needle approach to obtain blastomere biopsies from human preimplantation embryos for preimplantation genetic diagnosis (PGD). The method was first evaluated in a mouse system and shown to be compatible with a high degree of in vitro and in vivo development of biopsied mouse embryos. Furthermore, we showed that biopsied mouse embryos after transfer to recipient mice underwent implantation, normal development and delivery. Litters were followed through puberty and adulthood and shown to be normal with regard to sexual function and also a panel of biochemical and morphological parameters including organ histology. Successful human preimplantation diagnosis, followed by pregnancies and birth of healthy babies, was established with two out of three couples carrying a risk to transmit chromosomal abnormalities leading to severe disease. This is the first report of the successful use of a single-needle approach in human PGD. Considering its simplicity, we conclude that the single-needle approach is an attractive alternative for biopsies in PGD.

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%.

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.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.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 levelor = 150 IU/L 13-15 days after ET carried a pregnancy to term (the low risk group). Conversely, 50% of the patients with hCG150 IU/L carried a pathological pregnancy (the high risk group).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.

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.

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.

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.

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 obtamed from prepubertal rats primed with pregnant mare's serum gonadotropin. Oocytes were isolated and 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.

We studied the effect of low molecular weight fractions of human follicular fluid (FF1) on rat oocyte meiosis and progesterone secretion by the granulosa cells. Steroid-free FF1 extracts were filtered through Sephadex G50 gel. One highly retarded fraction was obtained (GF-3), which was either used for testing or further purified by filtration on Sephadex G10, the G10-3 fraction of which was used for experiments. The GF-3 and G10-3 fractions of FF1 inhibited (in a dose-dependent and reversible manner) meiosis of isolated rat oocytes during a 4-h culture. Similarly treated fractions of serum had no effect. The inhibition was not abolished by ether extraction, trypsin treatment, heating to 56 degrees C for 1 h or boiling for 5 min, whereas heating to 105 degrees C for 18 h decreased the effect. Gonadotropin-stimulated progesterone secretion by cumulus and mural granulosa cells was also dose-dependently and reversibly inhibited by the GF-3 fractions. Our results confirm those obtained in earlier studies on porcine oocyte maturation inhibitor (OMI).

Publisher Summary This chapter discusses acute stimulatory effects of luteinizing hormone-releasing hormone (LHRH) and LHRH analogues on the preovulatory rat follicle. In a study described in the chapter, immature Sprague–Dawley rats, 26 days old, were treated with 10 IU PMSG. This treatment resulted in the ovulation of 12–18 ova early on the morning of day 29. Ovarian follicles or granulosa cells were isolated on the morning of day 28. The follicles were incubated in Krebs–Ringer bicarbonate buffer with glucose and 1% bovine serum albumin. Granulosa cells were cultured in Eagle's minimal essential medium with Hepes and 10% fetal calf serum. For the in vivo experiments, the rats were hypophysectomized on the morning of day 28. It was found that mammalian oocytes remain arrested in the prophase stage of the first meiotic division until meiosis is resumed in the preovulatory follicle following the LH surge. Oocyte meiosis can also be initiated in vitro by the addition of gonadotrophin to isolated preovulatory rat follicles.

The effect of luteinizing hormone (LH) on the respiration of isolated cumulus cell complexes (the oocyte surrounded by cumulus granulosa cells) obtained from immature Sprague-Dawley rats injected with 10 IU pregnant mare's serum gonadotropin on day 30 was investigated. The cell complexes were isolated from preovulatory follicles of rats killed at specific time intervals on the day preceding ovulation, i.e., on day 32. The samples were incubated in Eagle's tissue culture medium. Oxygen uptake was recorded either with the Cartesian diver technique or with a recently described microspectrophotometric technique using hemoglobin as an indicator. Exposure to exogenous bovine LH in vitro or in vivo or to endogenous LH, i.e., the preovulatory LH-surge, resulted in a marked decrease in respiratory activity of the cumulus cell complex, as revealed by both techniques. The cumuli exposed to LH showed an oxygen uptake of approximately 40-65% of the control cumuli. The results suggest that LH has a direct effect on this cell complex resulting in a decreased oxidative metabolism.

Follicular oocytes were examined at various time intervals preceding ovulation in immature rats injected with 10 IU pregnant mare's serum goriadotropin (PMS) on the morning of day 30. A dose of 10 IU PMS was shown to give a physiological number of ova in the oviducts on the morning of day 33. Serum LH levels were determined with a radioimmunoassay and were found to increase on the afternoon of day 32. The earliest detectable change in the process of ovum maturation, viz. loss of the germinal vesicle and the nucleolus, occurred 2–3 hr after the increase in serum LH. This time relationship is very similar to that reported earlier for adult cycling rats. It is concluded that the PMS-injected immature rat is a suitable experimental animal for studies aimed to further elucidate the ovarian mechanisms triggering the resumption of the meiotic division of the follicular oocytes before ovulation. (Endocrinology 95: 1762, 1974)

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.

The effect of en-clomiphene and zu-clomiphene (10(-9)-10(-5) M) on progestin synthesis in cultured human granulosa cells was studied under basal conditions and in the presence of LH (100 ng/ml). Granulosa cells were obtained from either pre-ovulatory follicles of clomiphene-HMG-stimulated cycles or from large follicles of mid-to-late follicular phase of spontaneous cycles. The basal and LH-stimulated progesterone accumulation was dose-dependently reduced by en- and zu-clomiphene (10(-6)-10(-5) M) in cells from both groups studied, an effect similar to that of oestradiol. In contrast to oestradiol, both en- and zu-clomiphene (10(-6)-10(-5) M) reduced the basal and LH-stimulated pregnenolone accumulation in cells of stimulated cycles. The effect of clomiphene on progesterone, and pregnenolone accumulation was more pronounced in LH-stimulated cells than under basal conditions. There were no qualitative differences between the two isomers and the results were principally the same in cells from both groups of follicles studied. It is concluded that en-clomiphene and zu-clomiphene have similar inhibitory effects on progestin synthesis in human granulosa cells in vitro.

It has been shown earlier that LH decreases the oxygen consumption of the rat cumulus oophorus cells, i.e. the granulosa cells immediately surrounding the follicular oocyte. The decrease occurs in the preovulatory period at the time when the oocyte resumes and completes meiosis. This study examines in more detail the time-course of LH-induced inhibition of cumulus respiration. The results show that when isolated cumuli were exposed to LH during in vitro culture a significant inhibition of respiration occurred within one hour. When follicle-enclosed cumuli were exposed to LH, in vivo or in vitro, the decrease in respiration was not evident until 4 hours after the hormone exposure. By this time oocyte maturation was already in progress. The results suggest that the decrease in cumulus respiration may not be directly linked to the onset of oocyte meiosis.

Preovulatory follicles were isolated from immature rats 2 days after they were given a single injection of PMS gonadotrophin (PMSG). The follicles were either incubated intact or mechanically separated to obtain theca and granulosa cells. The samples were incubated for 2 h in a modified Krebs bicarbonate buffer containing albumin. The accumulation of progesterone, androstenedione, testosterone, and estradiol in the medium was determined by RIA. When the cells were isolated from rats killed before the LH surge, the intact follicles secreted predominantly estradiol in the hormone-free medium. The theca cells produced mainly androstenedione, and the granulosa cells produced mainly progesterone and estradiol. When LH was added to the incubation medium, the intact follicles increased the production of progesterone, androstenedione, and estradiol. In the theca cells, the in vitro addition of LH increased only the production of androstenedione while in the granulosa cells only progesterone was increased during t...

Preovulatory follicles were explanted on the day before ovulation from immature rats given a single injection of Pregnant Mare's Serum gonadotropin (PMS) 2 days earlier. The follicles were incubated for 4 h in modified Krebs bicarbonate buffer containing glucose and albumin in absence or presence of ovine luteinizing hormone (NIH-LH-S18; 0.1-10 mug/ml). The accumulation of progresterone, androstenedione and 17beta-estradiol in the medium was determined by radioimmunoassay. As in indicator of LH exposure the meiotic stage of the follicle-enclosed oocyte was determined at recovery by interference contrast microscopy. The first group of follicles were explanted in the morning, before the endogenous gonadotrophin surge. In hormone-free medium the oocytes remained in the dictyate stage, whereas addition of LH induced oocyte maturation. These follicles, when incubated in hormone-free medium, secreted predominantly androstenedione and estradiol and only low amounts of progesterone. In the presence of LH the secretion of all steroids was enhanced. The second group of follicles were explanted in the evening, 2-4 h after the endogenous gonadotrophin surge. After incubation in hormone-free medium the follicle-enclosed oocytes had matured. The steroid secretion by the follicles was different from that of the first group. In hormone-free medium they secreted predominantly progesterone and low amounts of androstenedione and estradiol. Addition of LH to the medium caused further enhancement of progesterone secretion, but had no effect on androstenedione and estradiol secretion. The third group of follicles were explanted in the evening from rats in which the preovulatory gonadotrophin surge had been prevented by Nembutal treatment. Oocyte maturation and steroid secretion did not differ from that found for the first group of follicles explanted in the morning. The results are compatible with the hypothesis that LH, after a transitory stimulation, inhibits androgen and estrogen secretion and stimulates progesterone secretion by the preovulatory ovarian follicle.

Graafian follicles were isolated by microdissection from the ovaries of PMS-injected immature rats killed at specific times on the day before ovulation. They were incubated in Krebs bicarbonate buffer containing 5 mm glucose and 1 % bovine serum albumin. The oocytes were recovered after incubation and examined by Nomarski interference contrast microscopy. The amount of lactate and glucose in the incubation medium was analysed enzymatically. Oocytes recovered from follicles extirpated in the morning, i. e. before the endogenous LH surge, and incubated for 2–10 h were in the dictyate stage, while addition of LH or FSH to the medium resulted in oocyte maturation as revealed by germinal vesicle breakdown (GVB) and polar body formation. The time-course of GVB in vitro was similar to that seen in vivo after exogenous LH. Both gonadotrophins markedly enhanced lactate accumulation in the medium and, as studied for LH, glucose uptake by the follicles. The effects of LH but not those of FSH, on GVB and lactate formation were prevented by the presence of an antiserum against the β-subunit of LH. The gonadotrophic effects on GVB could be mimicked by the addition of prostaglandin E2 to the medium. When the follicles were extirpated in the late afternoon, i. e. after the LH surge, and incubated in hormone-free medium for 4 h the oocytes showed GVB in a progressively increasing proportion depending on the time of follicle extirpation. Lactate formation by this group of follicles was markedly enhanced compared to that of "morning" follicles, but it could be even more stimulated by in vitro exposure to LH. A preliminary series of experiments on the effect of phosphodiesterase inhibitors showed that theophylline and isobutylmethylxanthine at certain concentrations completely blocked the LH effect on GVB, whereas a newly developed compound, ICI 63.197, in itself induced GVB.

In 28-day-old rats injected with PMSG, ovarian cyclic AMP and PGE and PGF increased 2 and 3-4 h respectively after the LH peak.