Evaluation of live birth rates and perinatal outcomes following two sequential vitrification/warming events at the zygote and blastocyst stages.

PURPOSE: To study the outcome of sequential cryopreservation-thawing of zygotes followed by the cryopreservation-thawing of blastocysts in the course of an IVF treatment on live birth rate and neonatal parameters. METHODS: Single center, retrospective chart review for the time period of 2015-2020. Clinical and perinatal outcomes were compared between frozen embryo transfer cycles utilizing twice-cryopreserved (n = 182) vs. once-cryopreserved (n = 282) embryos. Univariate and multivariable analyses were used to adjust for relevant confounders. RESULTS: After adjustment for maternal age, gravidity, parity, body mass index (BMI), paternal age, fertilization method used, the number of oocytes retrieved in the fresh cycle, fertilization rate, and transfer medium, the transfer of twice-cryopreserved embryos resulted in a reduced probability of live birth (OR, 0.52; 95% CI 0.27-0.97; p=0.041) compared to once-cryopreserved embryos. No differences in the sex ratio, the mean gestational age, the mean length at birth, or the mean birth weight were found between the two groups. CONCLUSION: The circumstantial use of sequential double vitrification-warming in course of treatment is associated with a reduced (but still reasonable) live birth rate compared to once-cryopreserved embryos. As the neonatal outcomes of twice-cryopreserved embryos are similar to once-cryopreserved embryos, this treatment option appears still valid as a rescue scenario in selected cases.

Dydrogesterone and 20α-dihydrodydrogesterone plasma levels on day of embryo transfer and clinical outcome in an anovulatory programmed frozen-thawed embryo transfer cycle: a prospective cohort study.

STUDY QUESTION: What are the plasma concentrations of dydrogesterone (DYD) and its metabolite, 20α-dihydrodydrogesterone (DHD), measured on day of embryo transfer (ET) in programmed anovulatory frozen embryo transfer (FET) cycles using 10 mg per os ter-in-die (tid) oral DYD, and what is the association of DYD and DHD levels with ongoing pregnancy rate? SUMMARY ANSWER: DYD and DHD plasma levels reach steady state by Day 3 of intake, are strongly correlated and vary considerably between and within individual subjects, women in the lowest quarter of DYD or DHD levels on day of FET have a reduced chance of an ongoing pregnancy. WHAT IS KNOWN ALREADY: DYD is an oral, systemic alternative to vaginal progesterone for luteal phase support. The DYD and DHD level necessary to sustain implantation, when no endogenous progesterone is present, remains unknown. While DYD is widely used in fresh IVF cycles, circulating concentrations of DYD and DHD and inter- and intraindividual variation of plasma levels versus successful treatment have never been explored as measurement of DYD and DHD is currently only feasible by high-sensitivity chromatographic techniques such as liquid chromatography/tandem mass spectroscopy (LC-MS/MS). STUDY DESIGN, SIZE, DURATION: Prospective, clinical cohort study (May 2018-November 2020) (NCT03507673); university IVF-center; women (n = 217) undergoing a programmed FET cycle with 2 mg oral estradiol (tid) and, for luteal support, 10 mg oral DYD (tid); main inclusion criteria: absence of ovulatory follicle and low serum progesterone on Days 12-15 of estradiol intake; serum and plasma samples were taken on day of FET and stored at -80°C for later analysis by LC-MS/MS; in 56 patients, two or more FET cycles in the same protocol were performed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women undergoing FET on Day 2 or Day 3 (D2, D3, cleavage) or Day 5 (D5, blastocyst) of embryonic development had blood sampling on the 3rd, 4th or 6th day of 10 mg (tid) DYD oral intake, respectively. The patient population was stratified by DYD and DHD plasma levels by percentiles (≤25th versus >25th) separately by day of ET. Ongoing pregnancy rates (a viable pregnancy at >10th gestational week) were compared between ≤25th percentile versus >25th percentile for DYD and DHD levels (adjusted for day of ET). Known predictors of outcome were screened for their effects in addition to DYD, while DYD was considered as log-concentration or dichotomized at the lower quartile. Repeated cycles were analyzed assuming some correlation between them for a given individual, namely by generalized estimating equations for prediction and generalized mixed models for an estimate of the variance component. MAIN RESULTS AND THE ROLE OF CHANCE: After exclusion of patients with 'escape ovulation' (n = 14, 6%), detected by the presence of progesterone in serum on day of ET, and patients with no results from LC-MS/MS analysis (n = 5), n = 41 observations for cleavage stage ETs and n = 157 for blastocyst transfers were analyzed. Median (quartiles) of plasma levels of DYD and DHD were 1.36 ng/ml (0.738 to 2.17 ng/ml) and 34.0 ng/ml (19.85 to 51.65 ng/ml) on Day 2 or 3 and 1.04 ng/ml (0.707 to 1.62 ng/ml) and 30.0 ng/ml (20.8 to 43.3 ng/ml) on Day 5, respectively, suggesting that steady-state is reached already on Day 3 of intake. DHD plasma levels very weakly associated with body weight and BMI (R2 < 0.05), DYD levels with body weight, but not BMI. Levels of DYD and DHD were strongly correlated (correlation coefficients 0.936 for D2/3 and 0.892 for D5, respectively). The 25th percentile of DYD and DHD levels were 0.71 ng/ml and 20.675 ng/ml on day of ET. The ongoing pregnancy rate was significantly reduced in patients in the lower quarter of DYD or DHD levels: ≤25th percentile DYD or DHD 3/49 (6%) and 4/49 (8%) versus >25th percentile DYD or DHD 42/149 (28%) and 41/149 (27%) (unadjusted difference -22% (CI: -31% to -10%) and -19% (CI: -29% to -7%), adjusted difference -22%, 95% CI: -32 to -12, P < 0.0001). LIMITATIONS, REASONS FOR CAUTION: Some inter- and intraindividual variations in DYD levels could be attributed to differences in time between last 10 mg DYD intake and blood sampling, as well as concomitant food intake, neither of which were registered in this study. Ninety percent of subjects were European-Caucasian and DYD/DHD blood concentrations should be replicated in other and larger populations. WIDER IMPLICATIONS OF THE FINDINGS: Daily 10 mg DYD (tid) in an artificial FET cycle is potentially a suboptimal dose for a proportion of the population. Measurement of DYD or DHD levels could be used interchangeably for future studies. The pharmacokinetics of oral DYD and associated reproductive pharmacodynamics need further study. STUDY FUNDING/COMPETING INTEREST(S): The trial was financed by university funds, except for the cost for plasma and serum sample handling, storage and shipment, as well as the liquid chromatography-mass spectrometry (LC-MS/MS) analysis of DYD, DHD and progesterone, which was financially supported by Abbott Products Operations AG (Allschwil, Switzerland). Abbott Products Operations AG had no influence on the study protocol, study conduct, data analysis or data interpretation. K.N. has received honoraria and/or non-financial support (e.g. travel cost compensation) from Ferring, Gedeon-Richter, Merck and MSD. A.M. has no competing interests. R.V. has no competing interests. M.D. has received honoraria and/or non-financial support from Ferring and Merck. A.S.-M. has no competing interests. T.K.E. has received honoraria and/or non-financial support from Roche, Novartis, Pfizer, Aristo Pharma, Merck. G.G. has received honoraria and/or non-financial support (e.g. travel cost compensation) from Abbott, Ferring, Gedeon Richter, Guerbet, Merck, Organon, MSD, ObsEva, PregLem, ReprodWissen GmbH, Vifor and Cooper. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov NCT03507673.

A randomised, multi-center, open trial comparing a semi-automated closed vitrification system with a manual open system in women undergoing IVF.

STUDY QUESTION: What are outcome and procedural differences when using the semi-automated closed Gavi® device versus the manual open Cryotop® method for vitrification of pronuclear (2PN) stage oocytes within an IVF program? SUMMARY ANSWER: A semi-automated closed vitrification method gives similar clinical results as compared to an exclusively manual, open system but higher procedure duration and less staff convenience. WHAT IS KNOWN ALREADY: A semi-automated closed vitrification device has been introduced to the market, however, little evaluation of its performance in a clinical setting has been conducted so far. STUDY DESIGN, SIZE, DURATION: This prospective, randomised, open non-inferiority trial was conducted at three German IVF centers (10/2017-12/2018). Randomization was performed on day of fertilization check, stratified by center and by indication for vitrification (surplus 2PN oocytes in the context of a fresh embryo transfer (ET) cycle or 'freeze-all' of 2PN oocytes). PARTICIPANT/MATERIAL, SETTING, METHODS: The study population included subfertile women, aged 18-40 years, undergoing IVF or ICSI treatment after ovarian stimulation, with 2PN oocytes available for vitrification. The primary outcome was survival rate of 2PN oocytes at first warming procedure in a subsequent cycle and non-inferiority of 2PN survival was to be declared if the lower bound 95% CI of the mean difference in survival rate excluded a difference larger than 9.5%; secondary, descriptive outcomes included embryo development, pregnancy and live birth rate, procedure time and staff convenience. MAIN RESULTS AND THE ROLE OF CHANCE: The randomised patient population consisted of 149 patients, and the per-protocol population (patients with warming of 2PN oocytes for culture and planned ET) was 118 patients. The survival rate was 94.0% (±13.5) and 96.7% (±9.7) in the Gavi® and the Cryotop® group (weighted mean difference -1.6%, 95% CI -4.7 to 1.4, P = 0.28), respectively, indicating non-inferiority of the Gavi® vitrification/warming method for the primary outcome. Embryo development and the proportion of top-quality embryos was similar in the two groups, as were the pregnancy and live birth rate. Mean total procedure duration (vitrification and warming) was higher in the Gavi® group (81 ± 39 min vs 47 ± 15 min, mean difference 34 min, 95% CI 19 to 48). Staff convenience assessed by eight operators in a questionnaire was lower for the Gavi® system. The majority of respondents preferred the Cryotop® method because of practicality issues. LIMITATIONS, REASON FOR CAUTION: The study was performed in centers with long experience of manual vitrification, and the relative performance of the Gavi® system as well as the staff convenience may be higher in settings with less experience in the manual procedure. Financial costs of the two procedures were not measured along the trial. WIDER IMPLICATIONS OF THE FINDINGS: With increasing requirements for standardization of procedures and tissue safety, a semi-automated closed vitrification method may constitute a suitable alternative technology to the established manual open vitrification method given the equivalent clinical outcomes demonstrated herein. STUDY FUNDING/COMPETING INTERESTS: The trial received no direct financial funding. The Gavi® instrument, Gavi® consumables and staff training were provided for free by the distributor (Merck, Darmstadt, Germany) during the study period. The manufacturer of the Gavi® instrument had no influence on study protocol, study conduct, data analysis, data interpretation or manuscript writing. J.H. has received honoraria and/or non-financial support from Ferring, Merck and Origio. G.G. has received honoraria and/or non-financial support from Abbott, Ferring, Finox, Gedeon Richter, Guerbet, Merck, MSD, ObsEva, PregLem, ReprodWissen GmbH and Theramex. The remaining authors have no competing interests. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov NCT03287479. TRIAL REGISTRATION DATE: 19 September 2017. DATE OF FIRST PATIENT'S ENROLMENT: 10 October 2017.

Characterization of early pregnancy placental progesterone production by use of dydrogesterone in programmed frozen-thawed embryo transfer cycles.

RESEARCH QUESTION: When and how does the gradual transition of the endocrine control of early pregnancy from the corpus luteum to the placenta, termed luteoplacental shift, take place? DESIGN: Prospective analysis of serum progesterone levels in pregnancies (n = 88) resulting from programmed frozen-thawed embryo transfer cycles in which ovulation was suppressed and no corpus luteum was present. Dydrogesterone, which does not cross-react with progesterone in immunoassay or spectrometric assay, was used for luteal phase and early pregnancy support. Progesterone, oestradiol and hCG were measured at regular intervals from before pregnancy achievement until +65 to 71 days after embryo transfer by Roche Elecsys electrochemiluminescence immunoassay (Elecsys ECLIA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: Serum progesterone remained at baseline levels on first blood analysis +9 to 15 days after embryo transfer and increased only marginally independently from the type of pregnancy up to +16 to 22 days after embryo transfer. From +23 to 29 days after embryo transfer, progesterone increased non-linearly above 1.0 ng/ml and increased further throughout the first trimester with elevated levels in multiples. Oestradiol levels increased in parallel with progesterone; hCG plateaued around +37 to 43 days. Progesterone levels were significant predictors for pregnancy viability from +23 to 29 days after embryo transfer onwards with best accuracy +37 to 43 days after embryo transfer (receiver operator characteristic analysis area under the curve 0.98; 95% CI 0.94 to 1; P = 0.0009). CONCLUSIONS: The onset of substantial progesterone production is the 7th gestational week. Progesterone increase is non-linear, depends on chorionicity and zygosity, and may have predictive potential on the outcome of pregnancies originating from frozen embryo transfer cycles.

Can a quality-of-life assessment assist in identifying women at risk of prematurely discontinuing IVF treatment? A prospective cohort study utilizing the FertiQoL questionnaire.

PURPOSE: This study aimed at assessing quality of life (QoL) by means of a validated measurement tool (FertiQoL) in German infertile patients before a first IVF/ICSI cycle with ancillary assessment of changes in FertiQoL scores after a failed first cycle and the predictive capacity of FertiQoL scores for treatment discontinuation. METHODS: The validated FertiQoL tool consisting of 24 questions regarding fertility-specific aspects of QoL was used for this prospective cohort study conducted at a university affiliated IVF center in Germany. Female patients (n = 119) filled out the FertiQoL form and questionnaire on sociodemographic variables on initiation of a first- and second-cycle IVF/ICSI treatment, respectively. RESULTS: On initiation of a first IVF/ICSI cycle, the mean scores (± standard deviation) for subscales emotional, mind-body, relational, and social items were 62 (± 19), 75 (± 17), 82 (± 13), and 78 (± 14), respectively; the total FertiQoL score was 73 (± 12). The mean total FertiQoL score at initiation of a first treatment cycle did not differ between patients who continued vs. discontinued treatment in case of no pregnancy achievement in the first cycle (73) (± 10) vs. 74 (± 14), p = 0.46). Furthermore, the mean total FertiQoL score did not change after an unsuccessful first IVF cycle (74 vs. 76, p = 0.46). CONCLUSIONS: There was no statistical difference in a small sample size for FertiQoL scores between all groups. In this study, FertiQoL scores were, therefore, not usable to predict withdrawal from infertility treatment.

What is the net effect of introducing vitrification for cryopreservation of surplus 2PN oocytes in an IVF program?

PURPOSE: The aim of this study was to accurately describe outcome differences (cryo-survival, pregnancy rate and live birth rate, both per ET and cumulatively), between the vitrification method and slow-freezing method of surplus 2PN oocytes in an IVF program. METHODS: In 2004, the freezing method for 2PN oocytes was changed from slow-cooling to vitrification. The data of 711 patients (timespan: 1/1999-7/2011; 410 vitrification and 301 slow-cooling events) undergoing a first IVF/ICSI cycles with freezing of 2PN oocytes were retrospectively analyzed. The outcome of one, the first, IVF cycle per patient was explored. The data were analyzed per freezing-thawing attempt as well as cumulatively per one complete IVF cycle, taking pregnancy occurrence after a fresh embryo transfer preceding the cryo-cycle(s) and other confounders (such as female age, elective vs. surplus 2PN cryopreservation) into account by means of exploratory regression analyses. RESULTS: In the vitrification and slow-cooling group, 756 and 376, respectively, attempts of thawing 2PN oocytes were recorded. Each attempt of thawing 2PN oocytes showed statistically significantly higher mean cryo-survival rates after vitrification (effect size approximately 30-40%, with vitrification cryo-survival consistently above 90% in all thawing attempts). Furthermore, the incidence of "zero survival" was lower after vitrification (0.5 vs. 7.3%, p < 0.01). It is estimated that the odds of achieving a live birth per one IVF cycle (fresh and frozen transfers combined) with vitrification of 2PN oocytes is increased approximately 1.4-fold (OR of 1.405, 95% CI 0.968-2.038; p = 0.07); however, statistical significance was not achieved due to sample size. Female age and elective cryopreservation of all 2PN oocytes without a fresh transfer (e.g., hyperresponders) were found to be negatively and positively, respectively, associated with the chance of achieving a live birth. CONCLUSIONS: The introduction of vitrification has a measurable impact on the efficacy of an IVF program. However, this effect is not large despite the impressively higher cryo-survival rates with vitrification. The "true" net efficacy effect of introducing 2PN vitrification in an IVF program will, in real life, be lower due to patients not having surplus 2PN oocytes available for freezing and later transfer.

Ovarian response to 150 µg corifollitropin alfa in a GnRH-antagonist multiple-dose protocol: a prospective cohort study.

The incidence of low (<6 oocytes) and high (>18 oocytes) ovarian response to 150 µg corifollitropin alfa in relation to anti-Müllerian hormone (AMH) and other biomarkers was studied in a multi-centre (n = 5), multi-national, prospective, investigator-initiated, observational cohort study. Infertile women (n = 212), body weight >60 kg, underwent controlled ovarian stimulation in a gonadotrophin-releasing hormone-antagonist multiple-dose protocol. Demographic, sonographic and endocrine parameters were prospectively assessed on cycle day 2 or 3 of a spontaneous menstruation before the administration of 150 µg corifollitropin alfa. Serum AMH showed the best correlation with the number of oocytes obtained among all predictor variables. In receiver-operating characteristic analysis, AMH at a threshold of 0.91 ng/ml showed a sensitivity of 82.4%, specificity of 82.4%, positive predictive value 52.9%and negative predictive value 95.1% for predicting low response (area under the curve [AUC], 95% CI; P-value: 0.853, 0.769-0.936; <0.0001). For predicting high response, the optimal threshold for AMH was 2.58 ng/ml, relating to a sensitivity of 80.0%, specificity 82.1%, positive predictive value 42.5% and negative predictive value 96.1% (AUC, 95% CI; P-value: 0.871, 0.787-0.955; <0.0001). In conclusion, patients with serum AMH concentrations between approximately 0.9 and 2.6 ng/ml were unlikely to show extremes of response.

Randomized, open trial comparing a modified double-lumen needle follicular flushing system with a single-lumen aspiration needle in IVF patients with poor ovarian response.

Study question: Is a modified double-lumen aspiration needle system with follicular flushing able to increase the mean oocyte yield by at least one in poor response IVF patients as compared to single-lumen needle aspiration without flushing? Summary answer: Follicular flushing with the modified flushing system did not increase the number of oocytes, but increased the procedure duration. What is known already: Most studies on follicular flushing were performed with conventional double-lumen needles in patients who were normal responders. Overall, these studies indicated no benefit of follicular flushing. Study design size, duration: Prospective, single-centre, randomized, controlled, open, superiority trial comparing the 17 G Steiner-Tan Needle® flushing system with a standard 17 G single-lumen aspiration needle (Gynetics®); time frame February 2015-March 2016. Participants/materials setting methods: Eighty IVF patients, 18-45 years, BMI >18 kg/m2 to <35 kg/m2, presenting with ≤ five follicles >10 mm in both ovaries at the end of the follicular phase were randomized to either aspirating and flushing each follicle 3× with the Steiner-Tan-Needle® automated flushing system (n = 40) or a conventional single-lumen needle aspiration (n = 40). Primary outcome was the number of cumulus-oocyte-complexes (COCs). Procedure duration, burden (Depression Anxiety and Stress Scale; DASS-21) and post-procedure pain were also assessed. Main results and the role of chance: Flushing was not superior with a mean (SD) number of COCs of 2.4 (2.0) and 3.1 (2.3) in the Steiner-Tan Needle® and in the Gynectics® group, respectively (mean difference -0.7, 95% CI: 0.3 to -1.6; P = 0.27). Likewise no differences were observed in metaphase II  oocytes, two pronuclear oocytes, number of patients having an embryo transfer and DASS 21 scores. The procedure duration was significantly 2-fold increased. Limitations reasons for caution: Testing for differences in the number of patients achieving an embryo transfer or differences in pregnancy rate would require a much larger sample size. Wider implications of the findings: The use of follicular flushing is unlikely to benefit the prognosis of patients with poor ovarian response. Study funding/competing interest(s): The Steiner-Tan Needles® and the flushing system were provided for free by the manufacturer. K.v.H. has received personal fees from Finox and non-financial support from Merck-Serono; M.D. has received personal fees from Finox and non-financial support from Merck-Serono. A.S.-M. has received personal fees and non-financial support from M.D., Ferring, Merck-Serono, Finox, TEVA. G.G. has received personal fees and non-financial support from M.D., Ferring, Merck-Serono, Finox, TEVA, IBSA, Glycotope, as well as personal fees from VitroLife, NMC Healthcare LLC, ReprodWissen LLC and ZIVA LLC. Trial registration number: NCT 02365350 (clinicaltrials.gov). Trial registration date: Sixth of February 2015. Date of first patient's enrolment: Ninth of February 2015.

Self-operated endovaginal telemonitoring: a prospective, clinical validation study.

OBJECTIVE: To study the comparability of self-operated endovaginal telemonitoring (SOET) with conventional two-dimensional transvaginal sonography (2D-TVS) monitoring during assisted reproductive technology (ART) cycles. DESIGN: Single center, observational, single-blinded cohort study. SETTING: University-affiliated in vitro fertilization center. PATIENT(S): A total of 60 women undergoing ART cycles. INTERVENTION(S): Explanation, training, and use of SOET system, and measurements of follicular and endometrial diameter with SOET and 2D-TVS. MAIN OUTCOME MEASURE(S): Correlation of the total number of follicles >10 mm measured by SOET versus conventional 2D-TVS. RESULT(S): In 16 cases (26.7%) the images were judged unsuitable for analysis. In these excluded cases the body mass index (BMI) was statistically significantly higher (29.3 vs. 24.4 kg/m(2)). The total number of follicles >10 mm was highly similar comparing SOET with conventional 2D-TVS (r = 0.91). For the concordance of whether more than 19 follicles or more than 25 follicles >10 mm were present, we found agreement between the methods in 43 of 44 cases (κ = 0.88) and 43 of 44 cases (κ = 0.85), respectively. For concordance on predefined human chorionic gonadotropin administration criteria, agreement was found in 39 of 44 cases (κ = 0.734). CONCLUSION(S): The incidence of SOET videos not suitable for analysis seems to be associated with higher BMI. Otherwise, SOET showed good agreement with conventional 2D-TVS both for follicles and endometrium measurements. More importantly we also found good concordance regarding the cutoffs relevant for clinical decisions.

Cumulative live birth rates after GnRH-agonist triggering of final oocyte maturation in patients at risk of OHSS: a prospective, clinical cohort study.

OBJECTIVES: To prospectively study the incidence of OHSS, live birth likelihood and neonatal outcome after GnRH-agonist triggering of final oocyte maturation and vitrification of all pronucleate (2PN) oocytes for later frozen-thawed embryo transfer (FRET) in an OHSS-risk population. STUDY DESIGN: Prospective, clinical cohort study (12/2004-5/2009). Forty patients undergoing ovarian stimulation in a GnRH-antagonist protocol and at risk of developing severe OHSS underwent triggering with 0.2mg triptorelin and elective vitrification of all 2PN-oocytes for later frozen-thawed embryo transfer. RESULTS: The incidence of OHSS was 0% (0/40; 95% confidence interval: 0.0-6.4%). Thirty-nine patients underwent 87 FRETs (mean number of FRETs per patient: 2.2+/-1.6; range: 1-7). The cumulative live birth rate per patient was 35.0% (14/40; 95% confidence interval: 23.9-48.0%). Mean time-to-conception resulting in live birth after agonist triggering was 24.2 (+/-17.1; range: 9-67) weeks. Nine healthy singletons and five twins were born. CONCLUSIONS: A treatment algorithm combining agonist trigger with vitrification of all 2PN-oocytes is feasible and safe, and provides patients with a good cumulative chance of live birth.

Ovarian hyperresponse to luteal phase GnRH-agonist administration.

INTRODUCTION: Herein we report a case of ovarian hyperresponse after luteal phase GnRH-agonist administration in a woman planning to undergo ovarian stimulation for IVF in a long GnRH-agonist protocol. MATERIALS AND METHODS: A normogonadotropic 25-year-old woman undergoing ICSI treatment for male factor infertility underwent three cycles of controlled ovarian stimulation, two in a GnRH-antagonist protocol, one in a long luteal GnRH-agonist protocol. RESULTS: In the first GnRH-antagonist cycle, ovarian stimulation was performed with 150 IE recombinant FSH and 22 oocytes were retrieved. In the second GnRH-antagonist cycle using the same protocol, six oocytes were retrieved. The estradiol levels on the day of hCG administration were 3,692 and 3,209 pg/ml, respectively. In a third cycle, 3.75 mg triptorelin was administered in the luteal phase and the patient showed ovarian hyperresponse to the endogenous gonadotropin flare with estradiol levels of 19,102 pg/ml, abdominal distension and discomfort, and massive bilateral ovarian enlargement (total ovarian volume 268 cm(3)). Ovarian cysts persisted for 4 weeks and necessitated cyst aspiration before further treatment. CONCLUSION: The flare-up effect of GnRH-agonist administration can, in rare cases, cause massive ovarian hyperresponse with associated health risks and significant postponement of treatment.

X-chromosome inactivation patterns and androgen receptor functionality influence phenotype and social characteristics as well as pharmacogenetics of testosterone therapy in Klinefelter patients.

Klinefelter syndrome is characterized by a vast range of phenotypes related to androgen effects. Testosterone (T) acts via the X-linked androgen receptor gene carrying the CAG repeat (CAGn) polymorphism, the length of which is inversely associated with androgen action and might account for the marked variation in phenotypes. In 77 newly diagnosed and untreated Klinefelter patients with a 47,XXY karyotype we assessed phenotype and social traits in relation to X-weighted biallelic CAGn length using X-chromosome inactivation analysis after digestion of leukocyte DNA with methylation-sensitive HpaII. Forty-eight men were hypogonadal and received T substitution therapy; in these, pharmacogenetic effects were investigated. The shorter CAGn allele was preferentially inactive. CAGn length was positively associated with body height. Bone density and the relation of arm span to body height were inversely related to CAGn length. The presence of long CAGn was predictive for gynecomastia and smaller testes, whereas short CAGn were associated with a stable partnership and professions requiring higher standards of education also when corrected for family background. There was a trend for men with longer CAGn to be diagnosed earlier in life. Under T substitution, men with shorter CAGn exhibited a more profound suppression of LH levels, augmented prostate growth, and higher hemoglobin concentrations. A significant genotype-phenotype association exists in Klinefelter patients: androgen effects on appearance and social characteristics are modulated by the androgen receptor CAGn polymorphism. The effects of T substitution are pharmacogenetically modified. This finding is magnified by preferential inactivation of the more functional short CAGn allele.

Lack of effect of a single i.v. dose of oxytocin on sperm output in severely oligozoospermic men.

BACKGROUND: ICSI into the oocyte is the only treatment currently available for most male patients with severe oligozoospermia who wish to father children. In order to perform ICSI, motile sperm need to be recovered from the ejaculate and, if no sperm or not enough motile sperm are recovered on the day of ICSI, testicular sperm extraction (TESE) must be performed. Oxytocin stimulates epididymal contractility and may be important for the release of stored sperm. The aim of this randomized single-blind cross-over study was to establish the effects of oxytocin on sperm output in severely oligozoospermic men. METHODS: Forty-nine infertile men with sperm concentrations <0.2 x 10(6)/ml were studied on two occasions after 3-4 days of sexual abstinence. They received an i.v. injection of saline or oxytocin 0.75 IU in random order, and commenced masturbation within 5 min. Ejaculate analysis was performed according to the WHO 1999 guidelines. RESULTS: A single i.v. dose of oxytocin resulted in no change in ejaculate volume (P = 0.4), total sperm count (P = 0.14) or sperm motility (P = 0.9). There was no significant correlation between the change in total sperm count and FSH levels (r = -0.32, P = 0.2), or the change in total sperm count and estradiol levels (r = -0.02, P = 0.9). Similar results were found in a subgroup of men with total sperm counts of <100. CONCLUSIONS: Our data indicate that a single-dose of i.v. oxytocin has no detectable effect on seminal parameters in men with severe oligozoospermia.

Prostate volume and growth in testosterone-substituted hypogonadal men are dependent on the CAG repeat polymorphism of the androgen receptor gene: a longitudinal pharmacogenetic study.

Testosterone (T) substitution in hypogonadal men results in growth of the prostate gland. T effects are mediated via the androgen receptor (AR). The length of the (CAG)n polymorphism of the AR gene is negatively associated with transcriptional activity and might account for variations in prostate growth during substitution therapy. In 131 hypogonadal men aged 18-69 yr, we assessed prostate volume longitudinally by transrectal ultrasonography and determined AR (CAG)n, sex hormone levels, and anthropometric measures. Sixty-nine men with primary and 62 with secondary hypogonadism began substitution therapy with im injections of T enanthate (n = 81), transdermal T preparations (n = 19), sc injections of human chorionic gonadotropin (n = 17), or oral T undecanoate (n = 14) for 2.4 +/- 0.8 yr. Average prostate size increased from 15.8 +/- 6.1 ml to 23.0 +/- 6.8 ml. ANOVA including covariates revealed initial prostate size to be dependent on age (P < 0.001) and baseline T levels (P = 0.01) but not on number of (CAG)n (ranging from 13-30; mean, 21.4 +/- 3.5). Prostate growth per year and absolute prostate size under substituted T levels (6.1 +/- 3.3 to 21.6 +/- 10.3 nmol/liter) were strongly dependent on (CAG)n, with lower treatment effects in longer repeats (both P < 0.001). Other significant predictors were initial prostate size (negative for growth rate and positive for absolute size) and age (positive for both growth rate and absolute size). The odds ratio for men with (CAG)n less than 20, compared with those with (CAG)n of 20 or more to develop a prostate size of at least 30 ml under T substitution, was 8.7 (95% confidence interval, 3.1-24.3; P < 0.001). This observation was strongly age dependent with a more pronounced odds ratio in men older than 40 yr. This first pharmacogenetic study on androgen substitution in hypogonadal men demonstrates a marked influence of the AR gene (CAG)n polymorphism on prostate growth.

Maintenance of spermatogenesis in hypogonadotropic hypogonadal men with human chorionic gonadotropin alone.

OBJECTIVE: It is generally accepted that both gonadotropins LH and FSH are necessary for initiation and maintenance of spermatogenesis. We investigated the relative importance of FSH for the maintenance of spermatogenesis in hypogonadotropic men. SUBJECTS AND METHODS: 13 patients with gonadotropin deficiency due to idiopathic hypogonadotropic hypogonadism (IHH), Kallmann syndrome or pituitary insufficiency were analyzed retrospectively. They had been treated with gonadotropin-releasing hormone (GnRH) (n=1) or human chorionic gonadotropin/human menopausal gonadotropin (hCG/hMG) (n=12) for induction of spermatogenesis. After successful induction of spermatogenesis they were treated with hCG alone for maintenance of secondary sex characteristics and in order to check whether sperm production could be maintained by hCG alone. Serum LH, FSH and testosterone levels, semen parameters and testicular Volume were determined every three to six Months. RESULTS: After spermatogenesis had been successfully induced by treatment with GnRH or hCG/hMG, hCG treatment alone continued for 3-24 Months. After 12 Months under hCG alone, sperm counts decreased gradually but remained present in all patients except one who became azoospermic. Testicular Volume decreased only slightly and reached 87% of the Volume achieved with hCG/hMG. During treatment with hCG alone, FSH and LH levels were suppressed to below the detection limit of the assay. CONCLUSION: Once spermatogenesis is induced in patients with secondary hypogonadism by GnRH or hCG/hMG treatment, it can be maintained in most of the patients qualitatively by hCG alone, in the absence of FSH, for extended periods. However, the decreasing sperm counts indicate that FSH is essential for maintenance of quantitatively normal spermatogenesis.