Investigation of luteal HCG supplementation in GnRH-agonist-triggered fresh embryo transfer cycles: a randomized controlled trial.

RESEARCH QUESTION: Does splitting the human chorionic gonadotrophin (HCG) support in IVF cycles triggered by a gonadotrophin-releasing hormone agonist result in a better progesterone profile? DESIGN: Randomized controlled three-arm study, performed at the Fertility Clinic, Odense University Hospital, Denmark. Patients with 12-25 follicles ≥12 mm were randomized into three groups: Group 1 - ovulation triggered with 6500 IU HCG; Group 2 - ovulation triggered with 0.5 mg GnRH agonist, followed by 1500 IU HCG on the day of oocyte retrieval (OCR); and Group 3 - ovulation triggered with 0.5 mg GnRH agonist, followed by 1000 IU HCG on the day of OCR and 500 IU HCG on OCR + 5. All groups received 180 mg vaginal progesterone. Progesterone concentrations were analysed in eight blood samples from each patient. RESULTS: Sixty-nine patients completed the study. Baseline and laboratory data were comparable. Progesterone concentration peaked on OCR + 4 in Groups 1 and 2, and peaked on OCR + 6 in Group 3. On OCR + 6, the progesterone concentration in Group 2 was significantly lower compared with Groups 1 and 3 (P = 0.003 and P < 0.001, respectively). On OCR + 8, the progesterone concentration in Group 3 was significantly higher compared with the other groups (both P<0.001). Progesterone concentrations were significantly higher in Group 3 from OCR + 6 until OCR + 14 compared with the other groups (all P ≤ 0.003). Four patients developed ovarian hyperstimulation syndrome in Group 3. CONCLUSION: Sequential HCG support after a GnRH agonist trigger provides a better progesterone concentration in the luteal phase.

Does the HCG trigger dose used for IVF impact luteal progesterone concentrations? a randomized controlled trial.

RESEARCH QUESTION: Is there an association between the ovulation trigger dose of human chorionic gonadotrophin (HCG) and endogenous progesterone production during the luteal phase? DESIGN: This randomized controlled four-arm study, at the Fertility Clinic, Odense University Hospital, Denmark, included women undergoing gonadotrophin-releasing hormone (GnRH) antagonist IVF treatment with ≤11 follicles ≥12 mm. Group 1-3 were triggered with 5000 IU, 6500 IU or 10,000 IU HCG, respectively, receiving 17α-hydroxyprogesterone caproate intramuscularly for luteal-phase support (LPS) to measure endogenous progesterone production. Group 4 received 6500 IU HCG trigger and vaginal progesterone. During the study, the 5000 IU and 10,000 IU HCG groups were switched from urinary to recombinant HCG, as urinary HCG was removed from market. Eight blood samples were drawn during the luteal phase. RESULTS: Ninety-four participants completed the study. There was a significant positive association between the HCG trigger dose and the progesterone at 8 days (P < 0.001), 10 days (P < 0.001) and 14 days (P < 0.001) post-oocyte retrieval. Comparing the groups individually revealed a significant difference in progesterone concentration between low and high trigger doses at 4 days (P = 0.037) and 8 days (P = 0.007) post-oocyte retrieval and between all intervention groups at oocyte retrieval + 6 days: group 1 and 2 (P = 0.011), group 2 and 3 (P = 0.042) and group 1 and 3 (P < 0.001). Higher HCG trigger dose increased the progesterone from the individual follicle. CONCLUSIONS: Increasing HCG trigger doses significantly increased endogenous progesterone concentration during the mid-late luteal phase.

The Danish National Register of assisted reproductive technology: content and research potentials.

The Danish National Register of assisted reproductive technology (ART) was initially established in 1994. The register comprises complete information on all ART procedures in public and private clinics in Denmark from 2013 and onwards, including baseline information on the cause of infertility and a number of health-related patient characteristics. The register enables monitoring and research on infertility treatment and reproductive topics in single women or couples seeking assisted reproduction, and the register is thus a key component of the Danish health information system within human reproduction. We aimed to provide an updated description of the register including advantages and pitfalls when using the register for reproductive epidemiological research, and a description of the accessibility for researchers. The Danish ART register is a valuable tool for epidemiological research. However, the inherent strengths and limitations ought to be in perspective when designing studies and interpreting the study results. Reports with annually aggregated data on ART treatments, can be accessed on the Danish Health Data Authority web page and researchers may obtain access to individual pseudonomized data via secure servers at the Danish Health Data Authority and Statistics Denmark.

Ovulation before or after intrauterine insemination does not affect live birth rates: a retrospective cohort study of 6701 cycles.

RESEARCH QUESTION: Should intrauterine insemination be carried out before or after follicle rupture, and is there a difference in sex ratio, according to follicle rupture at the time of insemination? DESIGN: In this retrospective cohort study conducted at the Fertility Clinic, Odense University Hospital, Denmark, data from 6701 homologous insemination cycles were analysed. Follicle rupture was determined by transvaginal ultrasonography at the time of insemination. The pregnancy rate, clinical pregnancy rate (CPR) and live birth rate (LBR) were recorded. RESULTS: In 2831 cycles (42.2%), follicle rupture had occurred at the time of insemination, whereas, in 3870 cycles (57.8%), no follicle rupture had occurred at the time of insemination. Overall, 1186 (17.7%) cycles resulted in a positive pregnancy test and no significant differences were found in pregnancy rate between rupture and no rupture of follicle (17.8% versus 17.7%, P = 0.90). Follicle rupture before or after insemination did not affect CPR (14.8% versus 15.0%, P = 0.86) or LBR (11.9% versus 12.2%, P = 0.75) per cycle. Moreover, the sex ratio of children born did not depend on follicle rupture (P = 0.20). After logistic regression with cluster and adjusting for baseline characteristics, no significant differences between groups were observed. CONCLUSION: Ovulation at the time of insemination is not associated with pregnancy rate, CPR, LBR or gender.

Mid-Luteal 17-OH Progesterone Levels in 614 Women Undergoing IVF-Treatment and Fresh Embryo Transfer-Daytime Variation and Impact on Live Birth Rates.

Introduction: Corpus luteum (CL) produces progesterone (P4) and 17-OH progesterone (17-OH P4) during the luteal phase. Contrary to P4, 17-OH P4 is not supplied as part of the luteal phase support following IVF-treatment. Therefore, measuring endogenous serum 17-OH P4 levels may more accurately reflect the CL function compared to monitoring serum P4 concentrations. Objective: To explore the correlation between mid-luteal serum 17-OH P4 levels and live birth rates and to explore the possible daytime variations in mid-luteal serum 17-OH P4. Design: Prospective cohort study. Patients: 614 women undergoing IVF-treatment and fresh embryo transfer. Intervention: All patients had serum 17-OH P4 measured 7 days after oocyte pick-up (OPU+7). Furthermore, on OPU+7, seven patients underwent repeated blood sampling during daytime to clarify the endogenous daytime secretory pattern of 17-OH P4. Outcome measure: Live birth rate. Secondary outcome measure: Daytime variation in serum 17-OH P4 levels. Results: The highest chance of a live birth was seen with mid-luteal 17-OH P4 between 6.0 and 14.0 nmol/l. The chance of a live birth was reduced below (RD -10%, p = 0.07), but also above the optimal range for 17-OH P4 (RD -12%, p = 0.04). Patients with diminished CL-function (17-OH P4 < 6 nmol/l) displayed clinically stable 17-OH P4 values, whereas patients with 17-OH P4 levels >6 nmol/l showed random 17-OH P4 fluctuations during daytime. Conclusion: The association between 17-OH P4 and reproductive outcomes is non-linear, and the negative effect of excessive CL-secretion seems to be just as strong as the negative effect of a reduced CL-function during the peri-implantation period.

A randomized controlled trial comparing the efficacy and safety of two HMG preparations gaining their LH bioactivity from different HCG sources.

In this prospective, controlled, randomized, multicentre, non-inferiority study, efficacy and safety of two HMG preparations (Menopur®- Ferring and Meriofert®- IBSA Institut Biochimique SA) for ovarian stimulation were compared (270 women undergoing IVF aged between 18 and 39 years; BMI 30 kg/m2 or less; less than three prior completed assisted reproduction technique cycles). A standard long down-regulation with gonadotrophin-releasing hormone agonist protocol, with HCG triggering was used; primary end-point was total number of oocytes retrieved; attention was paid toovarian hyperstimulation syndrome (OHSS). No statistically significant differences between the treatment groups were reported for most of the clinically significant end-points, including embryo quality, fertilization rate, implantation rate, ongoing pregnancy rate and live birth rate. Total number of oocytes retrieved was higher in the new HMG group compared with the reference (11.6 ± 6.6 and 9.7 ± 5.9, respectively, with a 95% CI of the difference equal +0.43 to +3.43). Increased number of oocytes was obtained through a shorter stimulation, but HMG units per oocyte retrieved were equivalent. The safety profile of the products for frequency of ovarian hyperstimulation syndrome was the same. This study showed that the new HMG preparation is a viable alternative for conducting ovarian stimulation in IVF cycles.

Costs of medically assisted reproduction treatment at specialized fertility clinics in the Danish public health care system: results from a 5-year follow-up cohort study.

OBJECTIVE: To examine the costs to the public health care system of couples in medically assisted reproduction. DESIGN: Longitudinal cohort study of infertile couples initiating medically assisted reproduction treatment. SETTING: Specialized public fertility clinics in Denmark. SAMPLE: Seven hundred and thirty-nine couples having no child at study entry and with data on kind of treatment and live birth (yes/no) for each treatment attempt at the specialized public fertility clinic. METHODS: Treatment data for medically assisted reproduction attempts conducted at the public fertility clinics were abstracted from medical records. Flow diagrams were drawn for different standard treatment cycles and direct costs at each stage in the flow charts were measured and valued by a bottom-up procedure. Indirect costs were distributed to each treatment cycle on the basis of number of visits as basis. Costs were adjusted to 2012 prices using a constructed medical price index. MAIN OUTCOME MEASURES: Live birth, costs. RESULTS: Total costs per live birth in 2012 prices were estimated to 10,755€. Costs per treated couple - irrespective of whether the treatment was terminated by a live birth or not - were estimated at 6607€. Costs per live birth of women <35 years at treatment initiation were 9338€ and 15,040€ for women ≥35 years. CONCLUSION: The public costs for live births after conception with medically assisted reproduction treatment are relatively modest. The results can be generalized to public fertility treatment in Denmark and to other public treatment settings with similar limitations in numbers of public treatment cycles offered.

A randomized clinical trial to evaluate the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) in embryo culture medium for in vitro fertilization.

OBJECTIVE: To evaluate the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) in embryo culture medium on ongoing implantation rate (OIR). DESIGN: Multicenter, randomized, placebo-controlled, double-blinded prospective design. SETTING: Fourteen Scandinavian fertility clinics. PATIENT(S): A total of 1,332 women with indication for in vitro fertilization or intracytoplasmic sperm injection; 1,149 received embryo transfer (GM-CSF: n = 564; control: n = 585). INTERVENTION(S): Oocytes were fertilized, and embryos cultured and transferred in control medium or test medium containing 2 ng/mL GM-CSF. MAIN OUTCOME MEASURE(S): OIR at gestational week 7, with follow-up at week 12 and birth. RESULT(S): At week 7, OIRs were 23.5% (GM-CSF), and 20.0% (control) (odds ratio [OR] 1.26, 95% confidence interval [CI] 0.91-1.75). At week 12, OIRs were 23.0% (GM-CSF) and 18.7% (control) (OR 1.35, 95% CI 1.06-1.72), and live birth rates were 28.9% and 24.1%, respectively (OR 1.35, 95% CI 1.03-1.78). The effect of GM-CSF was influenced by the human serum albumin concentration in the medium. Birth weight and abnormality incidence were similar in both groups. Exploratory analyses showed that GM-CSF increased OIR in women with previous miscarriage, especially in women with more than one miscarriage. CONCLUSION(S): Addition of GM-CSF to embryo culture medium elicits a significant increase in survival of transferred embryos to week 12 and live birth. Our results are consistent with a protective effect of GM-CSF on culture-induced embryo stress. GM-CSF may be particularly efficacious in women with previous miscarriage. CLINICAL TRIAL REGISTRATION NUMBER: NCT00565747.

Trends in rates of natural conceptions among Danish women born during 1960-1984.

STUDY QUESTION: The aim of the study was to analyse trends in the rate of natural conceptions (RNC) among birth cohorts of women born during the period 1960-1984. SUMMARY ANSWER: In this nationwide study of Danish-born female cohorts born during the period 1960-1984, we found a gradual decline in the RNC with successive birth cohorts. WHAT IS KNOWN ALREADY: Our results confirm the findings from a previous study on trends in RNC among native Danish women. STUDY DESIGN, SIZE, DURATION: This is a register-based cohort study. Our data set included 803 435 native Danish women born in 1960-1984, of whom 68.2% had conceived at least one child as of 1 January 2008, by which time the follow-up was terminated. PARTICIPANTS/MATERIALS, SETTING, METHODS: Data from Danish national registers were linked at the individual level using unique personal numbers assigned at birth to each resident. We analysed the data for the cohorts of native Danish women born in 1960-1984 and resident in Denmark in 2008. For these cohorts, we estimated the RNC per woman, defined as the mean number of live births minus live births after assisted reproductive technology (ART) plus the mean number of induced abortions. Births, abortions and births after ART were partly projected for the younger cohorts who had not finished their reproductive years before 2008. In addition, we looked at trends in hormonal contraception use. MAIN RESULTS: In the main projection scenario, the RNC gradually declined with successive cohorts from 2.39 among women born in 1960 to 2.15 among women born in 1984, with stable values of 2.15-2.16 projected in the youngest cohorts analysed, 1979-1984. The projected decline was a consequence of a decrease in induced abortion rates and an increase in the use of ART among the younger cohorts. Furthermore, we projected a cohort increase in the share of women without natural conceptions. LIMITATIONS, REASONS FOR CAUTION: A considerable portion of the results was based on projections, which involve uncertainty, especially concerning the results for women born in 1980 and later. In addition, information on IUI could not be included, which led to underestimation of the frequency of births after ART treatment. WIDER IMPLICATIONS OF THE FINDINGS: The results of our study contribute new insights to the research field of declining fertility rates in Europe and many other parts of the world.

Combination of IVF and IVM in naturally cycling women.

This study investigated the combination of an unstimulated IVF cycle with in-vitro maturation (IVM) of additional immature cumulus­oocyte­complexes (COC) from the same cycle collected at the same time as the spontaneous preovulatory follicle. This could potentially improve rates of embryo transfer and pregnancy/live births compared with conventional unstimulated IVF treatment and at the same time eliminate the risk of ovarian hyperstimulation syndrome. This prospective trial included 77 women with regular menstrual cycles. Age at inclusion was between 20 and 37 years. Results showed a retrieval rate of mature oocytes of 50/80 (62.5%) per cycle started and immature COC were collected in 74/80 (92.5%) cycles. The embryo transfer rate was 28/80 (35.0%) with mature oocytes and increased in total to 43/80 (53.8%) with IVM oocytes. Corresponding birth rates per transfer were 3/28 (10.7%) and 4/43 (9.3%). Birth rates per aspiration were 3/76 (3.9%) and 4/76 (5.3%). It is concluded that the protocol described here shows proof of concept, but the impact of the IVM procedure only reached a significant level regarding embryo transfer, not with live births. The reason for this is yet unclear, but asynchrony between endometrial factors and IVM oocytes together with unknown competence of IVM embryos is suspected.

Prenatal diagnostics in TESA/PESA pregnancies in Denmark 1995-2007: a shift from invasive procedures to nuchal translucency examination.

Evidently, children born after intracytoplasmic sperm injection (ICSI) are at an increased risk of having sex chromosomal abnormalities. Here we evaluate the change in methods used for prenatal diagnostics in patients having ICSI with epididymal or testicular sperm from the introduction of the procedure in 1995 until December 2007. Four hundred and fifty pregnancies resulted in the birth of 553 children. Of the Danish subpopulation 115 (34.2%) received nuchal translucency examination (NT) and 43 (12.8%) received invasive prenatal diagnostics (IPD). IPD was carried out in 11 out of 23 couples (48%) during the period 1995-1998. Since 2002, less than 10% chose to receive IPD. Twenty-one (57%) of 37 Danish women 37-44 years of age underwent IPD compared to only 22 (7.4%) of the 299 women less than 37 years of age (p < 0.001). Conversely, since 1999 the use of NT has gradually increased to a frequency of 88.9% in 2007. The partners of vasectomized men had significantly more often NT performed compared to those of non-vasectomized men. IPD were not otherwise associated with the etiology of azoospermia. This study documents a shift in prenatal diagnostics from IPD to NT for testicular sperm aspiration/percutaneous epididymal sperm aspiration (TESA/PESA) couples.

Derivation and characterisation of hESC lines from supernumerary embryos, experience from Odense, Denmark.

The derivation and characterisation of human embryonic stem cells provides a source of pluripotent stem cells with potential for clinical applications. Utilising locally sourced embryos from two IVF clinics, we derived and characterised five new cell lines for use in a non-clinical setting. Analysis of clinical data showed that the majority of embryos (94.5%) failed to reach the blastocyst stage of development and of all embryos, regardless of developmental status, 248 embryos were needed to create one stem cell line. From the number of embryos (69) which developed to the blastocyst stage 8.7% developed into cell lines. Using outgrowth of the whole blastocyst, we derived five new, unreported cell lines in Odense, Denmark between 2005 and 2006. Characterisation was carried out using RT-PCR, staining, karyotyping, EB formation and teratoma formation. The KMEB hESC lines will, in the future, be made available through the UK Stem Cell Bank (http://www.ukstemcellbank.org.uk/).

Register data on Assisted Reproductive Technology (ART) in Europe including a detailed description of ART in Denmark.

The European Society for Human Reproduction and Embryology (ESHRE) has monitored Assisted Reproduction in Europe since 1997. In 2001, 579 clinics from 23 countries reported 289,690 treatment cycles with: in vitro fertilization (IVF) 120,946, intracytoplasmatisk sperm injection (ICSI) 114,378, frozen embryo replacements (FER) 47,195 and egg donation (ED) 7,171. Intrauterine inseminations are less consistently reported, but in 2001, 15 countries reported 67,124 cycles (IUI-H 52 949 and IUI-D 14 185). Data from 2001 showed that in 12 countries, where all clinics reported the in vitro techniques to the register, 829 treatment cycles were performed per million inhabitants. The availability was highest in the Nordic countries (range 975-1,923 cycles). The proportion of infants born after ART with in vitro techniques ranged from 0.2% in Latvia to 3.9% in Denmark. In general, the impact of the in vitro techniques on the birth rate is highest in the Nordic countries (range 2.2-3.9%). Denmark is the only country with an established National ART reporting system involving all ART treatments both with the in vitro techniques and intrauterine inseminations. In the year 2002, a total of 20,837 treatment cycles were performed. The result was that 6.2% of all infants were born after assisted reproduction.

Impact of follicular-fluid meiosis-activating sterol in an albumin-based formulation on the incidence of human pre-embryos with chromosome abnormalities.

OBJECTIVE: To evaluate the effect of adding follicular-fluid meiosis-activating sterol (FF-MAS) in a novel 0.2% recombinant human albumin-based formulation to cumulus-enclosed oocytes on chromosomal status and development of pre-embryos. DESIGN: Multicenter, prospective, randomized, open (double-blind for vehicle and FF-MAS groups), four parallel groups, controlled trial. SETTING: Four public IVF clinics in Denmark. PATIENT(S): Two hundred eighteen women undergoing IVF donated 483 oocytes. INTERVENTION(S): Follicle-stimulating hormone/hCG-primed cumulus-enclosed oocytes randomized to 4 hours of exposure to medium with 1 or 10 micromol/L of FF-MAS dissolved in 0.2% recombinant human albumin, medium with 0.2% recombinant human albumin (vehicle control), or medium alone (control) before insemination. MAIN OUTCOME MEASURE(S): Primary endpoint: incidence of human pre-embryos with chromosomal abnormalities. Secondary endpoint: fertilization rate, cleavage rate, and pre-embryo quality assessed after 68 hours of culture. RESULT(S): At pre-embryo level, the overall abnormality rates in the control, vehicle control, and 1- and 10-micromol/L FF-MAS groups were 53%, 39%, 42%, 53%, respectively, and at blastomere level 49%, 44%, 44%, and 48%, respectively. After 20 and 26 hours, the fertilization rates were between 67% and 71% in all groups. No differences in the cleavage rates were observed. CONCLUSION(S): The concentrations of FF-MAS in a novel 0.2% recombinant human albumin-based formulation of FF-MAS did not increase the risk of chromosomal abnormalities in pre-embryos or blastomeres. No statistically significant differences in fertilization rate, cleavage rate, or number of good quality pre-embryos were found among the four groups.

A clinical study comparing PureSperm and SpermFilter for density gradient separation of human spermatozoa in assisted reproduction.

OBJECTIVE: To compare a new density gradient medium, SpermFilter, for purifying spermatozoa in assisted reproduction with the more established medium, PureSperm. DESIGN: Part 1, a multicenter study on 225 semen samples purified using either PureSperm (115 semen samples) or SpermFilter (110 semen samples). Part 2, a retrospective, single center study on a total of 898 assisted reproductive cycles (245 insemination cycles using husband semen, 58 insemination cycles using donor semen and 595 in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles. SETTING: Part 1, three fertility clinics in Denmark (two university-affiliated fertility clinics and one private clinic). Part 2, one university-affiliated fertility clinic in Denmark. MAIN OUTCOME PARAMETERS: Part 1, purity of purified spermatozoa (% motile), motility index and recovery of motile spermatozoa. Part 2, malformation and baby take-home rates (insemination cycles), fertilization, cleavage, implantation, malformation and baby take-home rates (IVF/ICSI cycles). RESULTS: No statistical differences were observed in any of the parameters investigated. CONCLUSION: SpermFilter is a valid alternative to PureSperm in assisted reproduction technology (ART).