Anti-Müllerian hormone remains highly expressed in human cumulus cells during the final stages of folliculogenesis.

This study evaluated whether anti-Müllerian hormone (AMH) was differentially expressed in cumulus (CC) and granulosa (GC) cells from large antral and pre-ovulatory follicles collected from individual follicles in women undergoing in-vitro maturation (IVM) or IVF treatment. Expression studies of AMH, AMH receptor 2, FSH receptor, aromatase and androgen receptor were performed in CC in IVM patients where cumulus-oocyte-complex had expanded, CC in IVM patients where cumulus-oocyte-complex remained compacted, GC from immature follicles and CC and GC from IVF patients. Microarray data on corresponding GC and CC from follicles from IVF patients was included. AMH expression was significantly higher in CC than in GC from both mature and immature follicles and in CC from immature follicles than in CC from pre-ovulatory follicles from IVF patients (P < 0.05). AMH expression was significantly higher in CC that remained compacted compared with those that had expanded (P < 0.008). AMH was correlated to the expression of FSH receptor, androgen receptor and AMH receptor 2 but not to aromatase expression. The expression pattern of AMH receptor 2 reflected that of AMH. AMH may exert intrafollicular functions even in human large antral and pre-ovulatory follicles and may be related to follicular health.

Concentrations of anti-Müllerian hormone in fluid from small human antral follicles show a negative correlation with CYP19 mRNA expression in the corresponding granulosa cells.

Ovaries surgically removed for fertility preservation from a total of 24 women served as a source of human small antral follicles, including the follicular fluid (FF) and the corresponding granulosa cells (GC). The FF was used to evaluate the intrafollicular concentrations of anti-Müllerian hormone (AMH), inhibin-B, estradiol, progesterone, androstenedione and testosterone. In GC mRNA expression of the AMH type II receptor (AMH-r2) was determined and correlated to the mRNA expression of CYP19 (aromatase), FSH-receptor (FSH-r) and LH-receptor (LH-r) and to the hormonal profiles of the corresponding FF. GC and FF from a total of 64 follicles (diameter of 3-9 mm) were evaluated. Concentrations of AMH in FF showed a highly significant inverse correlation with CYP19 mRNA expression in the corresponding GC and with concentrations of estradiol, progesterone and inhibin-B in the FF. However, a small subgroup of follicles exhibited high levels of AMH simultaneously with relative high levels of CYP19 mRNA. In contrast to AMH, mRNA expression of AMH-r2 was significantly positively correlated to the mRNA expression of FSH-r and CYP 19, but failed to correlate to any other measured parameters. These data confirms an intimate correlation between follicular AMH levels, AMH-r2, FSH-r expression and estradiol secretion in the developing human follicle.

De novo synthesis of placental protein-14 (PP14) and not PP12 by monolayer cultures of glandular epithelium of gestational endometrium.

Nine monolayer cell cultures of glandular epithelium from gestational endometrium were established from six apparently healthy women undergoing elective termination of pregnancy (7-11 weeks gestation). Radiolabel incorporation studies showed increasing incorporation of [35S]methionine into proteins present in supernatant and cytosol fractions over 48 h. The secreted proteins represented approximately 20% of the total incorporation of methionine into cytosolic proteins. De novo synthesis and secretion of placental protein-14 (PP14) and not PP12 was identified by a novel combination of line immunoelectrophoresis and autoradiography. All monolayer cultures demonstrated the presence of radiolabeled PP14, but not PP12, in the culture supernatants. These observations suggest that the glandular epithelial cells are the major site of synthesis and secretion of PP14 in human gestational endometrium.

Epidermal growth factor in small antral ovarian follicles of pregnant women.

Levels of epidermal growth factor (EGF) and steroids were measured by radioimmunoassay in follicular fluid (FF) aspirated from 114 small antral follicles with diameters from 1 to 6 mm and in serum from 19 women undergoing Caesarean section at term. Concentrations of EGF in FF were inversely and significantly correlated to follicular size, being 4.7 +/- 0.4 (mean +/- S.E.M.) nmol/l in follicles of 1-2 mm in diameter and declining to 2.2 +/- 0.2 and 1.4 +/- 0.2 nmol/l in follicles of 3-4 mm and 5-6 mm in diameter respectively. The mean +/- S.E.M. concentration of EGF in serum (0.7 +/- 0.03 nmol/l) was significantly lower than that in FF. Levels of EGF, progesterone and oestradiol in FF were not significantly correlated to one another. In contrast to EGF, levels of progesterone and oestradiol in FF did not vary significantly with follicular diameter in these small follicles. On the basis of these results we suggest that EGF is synthesized in small human antral follicles, and that EGF stimulates granulosa cell proliferation and follicle growth up to 6 mm in diameter. Furthermore, the high intrafollicular levels of EGF may protect the small follicles against untimely effects of high levels of FSH, for instance during the mid-cycle surge of gonadotrophins. It is concluded that EGF plays an important role as an autocrine and/or paracrine regulator of development of small antral follicles in women.

Submucous paracervical blockade compared with intramuscular meperidine as analgesia during labor: a double-blind study.

A double-blind study was carried out to compare the effect of submucous paracervical blockade using 12 mL 0.25% bupivacaine (55 women) to the effect of intramuscular injection of 75 mg meperidine (62 women) during the first stage of labor. All 117 were normal primiparous pregnancies. Seventy-eight percent of the women in the paracervical blockade group achieved full or acceptable pain relief against 31% in the meperidine group (P less than .01). Transient fetal bradycardia occurred in two cases in the paracervical blockade group and one in the meperidine group; all infants were born in good condition. Fetal distress, defined as an umbilical artery pH of 7.15 or less and/or a one-minute Apgar score of 7 or less was more frequent in the meperidine group (16 infants) than in the paracervical blockade group (six infants) (P less than .05). Submucous paracervical blockade is superior to intramuscular meperidine as pain relief during labor. Furthermore, meperidine results in more infants with asphyxia as compared with paracervical blockade.

No evidence for decreasing semen quality in four birth cohorts of 1,055 Danish men born between 1950 and 1970.

OBJECTIVE: To compare the quality of semen in 1,055 Danish men born between 1950 and 1970 who are assumed to represent a random sample of the Danish male population of fertile age. DESIGN: Retrospective review of data on semen quality at the time of the female partner's first IVF treatment. SETTING: The Fertility Clinic, Odense University Hospital, Odense, Denmark. PATIENT(S): One thousand fifty-five male partners of women with tubal infertility who were referred for IVF treatment consecutively during the period 1990-1996. INTERVENTION(S): Analysis of the semen samples delivered and used in connection with the couples' first IVF treatment. MAIN OUTCOME MEASURE(S): Year of birth, age at time of sample collection, sperm concentration, and semen volume. RESULT(S): The mean sperm concentration (+/- SD) was 183.7 x 10(6) mL and the mean semen volume (+/- SD) was 3.9 mL. A considerable variation in both parameters was found from year to year, but no significant change occurred in either parameter throughout the entire period. When four birth cohorts were compared, a later year of birth was not associated with any change in sperm concentration or semen volume. CONCLUSION(S): Sperm concentration and semen volume were not related to year of birth, indicating that sperm quality has not changed in the Danish male population during the last 20-30 years.

Human menopausal gonadotropin versus recombinant follicle-stimulating hormone in normogonadotropic women down-regulated with a gonadotropin-releasing hormone agonist who were undergoing in vitro fertilization and intracytoplasmic sperm injection: a prospective randomized study.

OBJECTIVE: To evaluate clinical and endocrinological effects of intranasal (IN) vs. subcutaneous (SC) GnRH-a for pituitary down-regulation combined with hMG vs. rFSH. DESIGN: Prospective, randomized study. SETTING: University hospital, IVF unit. PATIENT(S): Three hundred seventy-nine normogonadotropic women eligible for IVF or ICSI. INTERVENTION(S): Randomization to intranasal (IN) or SC GnRH-a and to hMG or rFSH. MAIN OUTCOME MEASURE(S): Oocytes retrieved, embryos developed, clinical pregnancy, and delivery rates. Serum hormone concentrations on stimulation days 1 (S1) and 8 (S8), and oocyte pick-up (OPU) day. RESULT(S): After randomization, four groups were formed: IN/hMG (n = 100), IN/FSH (n = 98), SC/hMG (n = 89), and SC/FSH (n = 92). Mean number of oocytes retrieved and of transferable and transferred embryos were similar in the four groups. Clinical pregnancy rate per started cycle was significantly higher in the IN/HMG group than in the SC/FSH group (P<.05) and was intermediate in the two remaining groups. Se-LH on S8 in the two SC groups was significantly lower than in the two IN groups. Se-E2 on S8 in the SC/FSH group was significantly lower than in the other three groups. CONCLUSION(S): The clinical and endocrinological outcome in IVF and ICSI-treated normogonadotropic women is significantly influenced by mode of down-regulation as well as gonadotropin formulation.

Developments in drugs for ovarian stimulation.

Drugs for ovarian stimulation have been improved during the last decades. Initially gonadotrophins were extracted from human pituitary glands and urine; nowadays they are produced from transformed cell-lines. All three gonadotrophins--follicle stimulating hormone (FSH), luteinizing hormone (LH) and human chorionic gonadotrophin (hCG)--are now marketed as recombinant (r-) products. The near-100% pure FSH preparations might, in some situations, cause abnormally low LH levels and it is likely that the addition of LH may be beneficial in these situations. It is possible that r-LH will become available in sufficient dosages to replace hCG for ovulation induction and this may reduce the incidence of ovarian hyperstimulation syndrome due to its shorter half-life. In parallel to the development of gonadotrophin preparations, protocols for ovarian stimulation are now more comfortable for the patients, especially with the introduction of gonadotrophin receptor hormone (GnRH)-agonists in the early 1980s and, more recently, the introduction of GnRH-antagonists.

Human menopausal gonadotropin versus recombinant follicle stimulation hormone for ovarian stimulation in assisted reproductive cycles.

BACKGROUND: hMG and recombinant FSH, have both been used successfully for controlled ovarian hyperstimulation in in vitro fertilization and embryo transfer (IVF-ET). OBJECTIVES: To compare the effectiveness of hMG with rFSH in ovarian stimulation protocols in IVF or ICSI treatment cycles. SEARCH STRATEGY: We searched the Cochrane Menstrual Disorders and Subfertility Group trials register (searched 3rd Jan 2002), PubMed, MEDLINE, Web of Science (all searched 1985 to May 15 2002), and reference lists of articles. We also contacted manufacturers and researchers in the field. SELECTION CRITERIA: Randomised trials comparing hMG with rFSH for ovarian stimulation in IVF or ICSI treatment for treatment of infertility in normogonadotrophic women. DATA COLLECTION AND ANALYSIS: The main outcome measure was ongoing pregnancy/live birth per woman. Secondary outcomes included total gonadotrophin dose used, cancellation, number of oocytes retrieved, implantation, clinical pregnancy per woman, multiple pregnancy, spontaneous abortion and ovarian hyperstimulation syndrome. Peto odds ratios (OR) for hMG relative to rFSH were calculated after testing for homogeneity of treatment effect across all trials. Analyses were performed separately for the three different GnRHa protocols used: (1) without GnRHa down-regulation, (2) with GnRHa down-regulation using a short protocol and (3) with GnRHa down-regulation using a long protocol. MAIN RESULTS: Eight trials that met the inclusion criteria could be identified. One trial did not use down-regulation, one trial used a short protocol and six trials used a long down-regulation protocol. In the one trial with non-down-regulated women and in the one trial that used a short down-regulation protocol there was no evidence of a difference between hMG and rFSH in any clinical outcome. Data of four truly randomised trials in women down-regulated using a long protocol could be pooled. There was no evidence of a difference between hMG and rFSH in ongoing pregnancy/live birth per woman (OR 1.27; 95% CI 0.98 to 1.64). Furthermore there was no clear difference on any of the secondary outcomes, although the clinical pregnancy rate per woman was of borderline significance in favour of hMG (summary OR 1.28; 95% CI 1.00 to 1.64). The other secondary outcomes were comparable for both gonadotrophins. REVIEWER'S CONCLUSIONS: For all three GnRHa protocols analysed there is insufficient evidence of a difference between hMG and rFSH on ongoing pregnancy or live birth. More large randomised trials are needed to estimate the difference between hMG and rFSH more precisely. Such trials should preferably (1) use a consistent long GnRHa protocol and (2) use a fixed dose of gonadotrophin such to prevent potentially subjective decisions of the clinician in dosing and (3) take live birth as primary endpoint. At this moment in time however, in prescribing gonadotrophins for ovarian hyperstimulation in IVF one should use the least expensive medication.

[Intrauterine and intracervical insemination with frozen donor semen. A comparative study]. / Intrauterin og intracervikal insemination med frossen donorsaed. En sammenlignende undersøgelse.

Intracervical insemination (ICI) with cryopreserved donor semen was performed in 38 women. These inseminations were performed about the time of ovulation twice per cycle in a total of 215 cycles during the period 1.1.1989 to 31.12.1990. Nine of these women became pregnant corresponding to 4% (9/215) per insemination cycle. From 1.12.1989, intrauterine insemination was introduced (IUC) with swim-up/wash-prepared cryopreserved donor semen prepared in the department. Twenty-two women who had undergone from three to 12 (average 6.1) ICI treatment cyclic without resulting pregnancy were submitted to IUI with cryopreserved donor semen at the time of ovulation once per cycle for a total of 32 cycles during the period 1.12.1989 to 31.12.1990. Ten of these (45%) became pregnant corresponding to 31% per insemination cycle. The reason for this significant improvement in the results as compared with ICI is to be found not only in alterations in the semen preparations and the technique of insemination but also in consequent employment of hormone stimulation and meticulous monitoring of the cycles. Employment of the described therapeutic schema for "programmed cycles" is recommended in order to minimize the need for resource-demanding cycle monitoring and to avoid weekend work.

[Preimplantation genetic diagnosis. Technical and ethical aspects and international experiences]. / Praeimplantations genetisk diagnostik.

In vitro fertilisation (IVF) is now an established treatment for infertility, and therefore preimplantation genetic diagnosis (PGD) has become an attractive diagnostic possibility on embryos at risk of having serious genetic and chromosomal abnormalities. PGD enables screening of certain genetic and chromosomal abnormalities before a pregnancy is established. In Denmark, The Minister of Health has in a recently proposed law on assisted reproduction recommended the implementation of pre-implantation genetic diagnosis on pre-embryos at risk of having serious genetic disorders. The aim of this review is to summarize international experience with the method and discuss its advantages and disadvantages. We conclude, that it is too early to implement the method as a routine clinical diagnostic analysis in Denmark, and that further research on the reliability of the method, the economy as well as other alternatives is recommended.

[The effectiveness of in vitro fertilization. A cohort study of 300 couples]. / In vitro-fertiliseringsbehandlingens effektivitet. En kohorteundersøgelse af 300 par.

A cohort of 300 couples, starting their first in-vitro fertilization (IVF) attempt between 1990 and 1992, were followed until completion of their treatment. A total of 897 treatment cycles were initiated. Of these 213 (23.7%) were cancelled, giving a total of 684 embryo replacements. One hundred and forty-one (47%) couples completed their treatment with delivery of at least one healthy baby. Ninety-eight (31.3%) couples completed their treatment without achieving delivery of a living baby, and 40 (13.7%) couples cancelled their treatment due to varying reasons. The treatment in seven couples was cancelled before completion due to medical reasons. In 12 women no transfer took place. IVF treatment is an effective procedure giving a "baby take home rate" of 47% within one to three cycles. A cohort study gives a realistic view of the probability of childbirth.

[Fertilization in vitro. A review over the medical indications and suggestion to unified guidelines for public fertility clinics in Denmark]. / In vitro fertilisering. En oversigt over medicinske indikationer og forslag til foelles retningslinier ved de offentlige danske fertilitetsklinikker.

Medical indications for in vitro fertilization and embryo transfer (IVF-ET) internationally and in Denmark are reviewed. Reports from large international centres document that tubal infertility, unexplained infertility, endometriosis and male infertility are equally good indications for IVF. Traditionally, tubal infertility has been the only medical indication qualifying for IVF treatment within the National Health Service in Denmark. Thus, in this country, couples with unexplained and male infertility and with endometriosis have to pay up to 25,000 D.Kr. per IVF-ET treatment in private fertility clinics. Since there is no scientific basis for this discrimination, it is urged that the present rules are changed, so that couples with unexplained and male infertility and endometriosis are also allowed IVF treatment free of charge in the public fertility clinics.

[Ovum donation. A review of and a suggestion to unified guidelines for treatment at public fertility clinics in Denmark]. / Aegdonation. En oversigt samt forslag til foelles retningslinier for behandling ved de offentlige danske fertilitetsklinikker.

The most common indication for oocyte donation is ovarian insufficiency due to premature menopause or resistant ovarian syndrome and ovarian dysgenesis with either normal or abnormal (e.g. Turner's syndrome) karyotype. In Denmark, oocyte donation must be anonymous, and the donors have to be other infertile patients undergoing in vitro fertilisation (IVF), treatment. It is suggested, that the National Health Service offers oocyte donation to hypergonadotropic women with ovarian insufficiency, as well as to a few other groups who fulfil the criteria for IVF treatment, but where this treatment cannot be completed. Oocyte donors must be less than 35 years old, physically and mentally healthy and without major genetic diseases in the family. The donor must be screened for HIV, hepatitis, syphilis, chlamydia and gonorrhoea. We propose that those patients who have more than six oocytes aspirated, allowing "surplus" oocytes to be donated. It is also proposed that the departmental order from the Ministry of Health be changed, so that normally fertile women are allowed to donate oocytes. Oocyte donation should be reported to a central authority.

[Freezing of human embryos. A review of and a suggestion to unified guidelines for treatment at public fertility clinics in Denmark]. / Nedfrysning af menneskelige aeg. En oversigt samt forslag til faelles retningslinier for behandling ved de offentlige danske fertilitetsklinikker.

In Denmark, legislation has made cryopreservation of human oocytes and embryos possible since October 1992. The legislation is reviewed. Cryopreservation of embryos constitutes a significant improvement of infertility treatment. The number of oocyte pick-ups and the number of embryos transferred can be reduced without compromising the total likelihood of success of in-vitro fertilization (IVF) treatment. According to Danish law, frozen embryos can only be stored for one year. This limit will interfere with patient expectations in numerous cases, and the time limit should be expanded. Moreover, freezing of embryos should be allowed in connection with oocyte donation programmes.

Number of germ cells and somatic cells in human fetal ovaries during the first weeks after sex differentiation.

BACKGROUND: This study presents the number of germ cells and somatic cells in human fetal ovaries during week 6 to week 9 post conception, i.e. the first weeks following sex differentiation of the gonads. METHODS: One ovary with attached mesonephros from each of 11 individual legal abortions was used for estimation of cell numbers. After recovery of the fetus, the ovary-mesonephric complexes were immediately isolated, fixed and processed for histology. A stereological method was utilized to estimate the total number of oogonia in all ovaries and somatic cells in seven of them. RESULTS: The number of oogonia per ovary increased from approximately 26,000 in week 6 to approximately 250,000 in week 9 and somatic cells from approximately 240,000 to approximately 1.4 x 10(6). The ratio of oogonia to somatic cells tended to increase throughout the period. The concentration of oogonia was similar in the cranial (mesonephric connected) part and the caudal part of the ovaries. CONCLUSIONS: This is the first stereological estimation of the number of oogonia and somatic cells in human fetal ovaries, and the first estimation of germ cells and somatic cells in ovaries aged <9 weeks. The number of oogonia in week 9 is comparable to the numbers previously published based on non-stereological estimations. We found early stages of meiosis in fetal ovaries from week 9.

Hormonal characteristics of follicular fluid from women receiving either GnRH agonist or hCG for ovulation induction.

BACKGROUND: A recent prospective randomized study from our group compared GnRH agonist (0.5 mg buserelin) and hCG (10,000 IU) for triggering of ovulation following a flexible antagonist protocol. The agonist group showed a poor reproductive outcome despite luteal phase support with progesterone and estradiol (E(2)). In the present prospective observational study, the health status of follicles from the above study was monitored by analysing the hormonal content of frozen/thawed follicular fluid samples. The aim was to test whether the poor reproductive outcome could be related to a defective pre-ovulatory follicular maturation resulting in oocytes with a compromised developmental competence. METHODS: Hormone concentrations were measured in two individual follicular fluid samples from each of 32 women receiving buserelin and 37 receiving hCG, thus representing a subset of the follicles retrieved. RESULTS: Follicular fluid levels of LH in the agonist group as compared with the hCG group was 11.1 +/- 0.5 versus 3.6 +/- 0.3 IU/l (mean +/- SEM; P < 0.001); FSH, 6.3 +/- 0.6 versus 3.3 +/- 0.2 IU/l (P < 0.001); hCG, not determined versus 139+/-8 IU/l; E(2), 1.9 +/- 0.2 versus 1.8 +/- 0.2 micromol/l (P > 0.10); progesterone, 70 +/- 4 versus 93 +/- 6 micromol/l (P < 0.001); inhibin-A, 36.9 +/- 3.1 versus 37.1 +/- 2.5 ng/ml (P > 0.10) and inhibin-B, 35.6 +/- 2.8 versus 40.1 +/- 3.1 ng/ml (P > 0.10). Thus, pronounced hormonal differences exist in follicular fluid, and the collective concentration of all three gonadotropins and the follicular fluid concentration of progesterone were much higher in the group of women receiving hCG for ovulation induction. CONCLUSION: The study suggests that GnRH agonist results in proper pre-ovulatory follicular maturation, but the ovulatory signal--probably in synergy with the resulting pituitary down-regulation--is too low to support appropriate corpus luteum (CL) function.

Epidermal growth factor (EGF) in human preovulatory follicles.

Concentrations of epidermal growth factor (EGF), progesterone (P4) and oestradiol (E2) were determined in follicular fluid (FF) aspirated from 69 preovulatory follicles in 15 women undergoing ovarian hyperstimulation and ovulation induction as part of IVF-ET treatment for infertility. Sixty-nine oocytes were obtained from these FF aspirates and 35 (51%) fertilized and cleaved in vitro. Pregnancy was achieved in five (33%) of the women. Concentrations of EGF in FF ranged between 0.60 and 2.42 ng/ml, corresponding to approximately 50% of the level in serum at the time of follicle aspiration. A significant linear correlation between levels of EGF in FF and in serum was demonstrated. By contrast, no significant relationships were found between intrafollicular levels of EGF and those of P4 and E2, or between EGF in FF and oocyte cleavage rate in vitro and the achievement of pregnancy after ET. It is concluded that EGF in FF of the human preovulatory follicles reflects a passive diffusion into the follicle from the circulation. Moreover, that intrafollicular EGF is not involved in the local regulation of human preovulatory follicle and oocyte maturation. These results, however, do not preclude an auto- and/or paracrine role of intrafollicular EGF in smaller human follicles.

Increased risk of early pregnancy loss by profound suppression of luteinizing hormone during ovarian stimulation in normogonadotrophic women undergoing assisted reproduction.

The impact of suppressed concentrations of circulating luteinizing hormone (LH) during ovarian stimulation on the outcome of in-vitro fertilization or intracytoplasmic sperm injection treatment in 200 consecutive, normogonadotrophic women (couples) was analysed retrospectively. A standard stimulation protocol with mid-luteal gonadotrophin-releasing hormone (GnRH) agonist down-regulation and ovarian stimulation with recombinant follicle stimulating hormone (FSH) was used in all cases. Blood was sampled from each woman on stimulation days 1 and 8 for analysis of oestradiol and LH in serum. A threshold value of serum LH of 0.5 IU/l on stimulation day 8 (S8) was chosen to discriminate between women with low or 'normal' LH concentrations. Low concentrations of LH on S8 (<0.5 IU/l) were found in 49% (98/200) of the women. This group of women was comparable with the normal LH group with regard to pre-treatment clinical parameters, and to the parameters characterizing the stimulation protocol with the exception of serum oestradiol concentration, which on S8 was significantly lower than in the normal LH group (P < 0.001). The proportion of positive pregnancy tests was similar in the two groups (30% versus 34% per started cycle), but the final clinical treatment outcome was significantly different, with a five-fold higher risk of early pregnancy loss (45% versus 9%; P < 0.005) in the low LH group and consequently a significantly poorer chance of delivery than in the normal LH group. It is concluded that a substantial proportion of normogonadotrophic women treated with GnRH agonist down-regulation in combination with FSH, devoid of LH activity, experience LH suppression, which compromises the treatment outcome. Whether these women would benefit from supplementation with recombinant LH or human menopausal gonadotrophin during ovarian stimulation, remains to be proven in the future by prospective randomized trials.