Prediction of the lower serum anti-Müllerian hormone threshold for ovarian stimulation prior to in-vitro fertilization using the Elecsys® AMH assay: a prospective observational study.

BACKGROUND: In assisted reproductive technology, prediction of treatment failure remains a great challenge. The development of more sensitive assays for measuring anti-Müllerian hormone (AMH) has allowed for the possibility to investigate if a lower threshold of AMH can be established predicting very limited or no response to maximal ovarian stimulation. METHODS: A prospective observational multicenter study of 107 women, < 40 years of age with regular menstrual cycle and serum AMH levels ≤ 12 pmol/L, treated with 300 IU/day of HP-hMG in a GnRH-antagonist protocol. AMH was measured before treatment start using the Elecsys® AMH assay by Roche Diagnostics. The ability of AMH to predict follicular development and ovarian response was assessed by receiver operating characteristics (ROC). Furthermore, the relationship between AMH at start of stimulation and cycle outcome was investigated using multivariate logistic regression analysis. RESULTS: Five out of 107 cycles (4.7%) were cancelled due to lack of follicular development and 60/107 (56%) women did not reach the classical hCG criteria for ovulation induction (≥ 3 follicles of ≥17 mm). An AMH threshold of 4 pmol/L predicted failure to reach the classical hCG criteria with 89% specificity and 53% sensitivity and an area under the curve (AUC) of 0.76 (95% CI 0.66-0.85). AMH predicted cycle cancellation due to lack of follicular development, using a cut-off value of 1.5 pmol/L, with a specificity of 96% and sensitivity of 80% (AUC = 0.92, 95% CI 0.79-1.00). A single-unit increase in AMH was associated with a 29% decrease in odds of failure to reach the classical hCG criteria (OR 0.71 95% CI 0.59-0.85, p < 0.01). The lowest AMH value compatible with a live birth was 1.3 pmol/L. CONCLUSIONS: Among women with a limited ovarian reserve, pre-treatment serum AMH levels significantly predicted failure to reach the classical hCG triggering criteria and predicted lack of follicular development using a new sensitive assay, but AMH was not suitable for withholding fertility treatment, as even very low levels were associated with live births. TRIAL REGISTRATION: Not relevant.

Gonadotropin receptor variants are linked to cumulative live birth rate after in vitro fertilization.

PURPOSE: The objective was to investigate if the gonadotropin receptor variants N680S (N: asparagine, S: serine, rs6166) in the follicle-stimulating hormone receptor (FSHR) and N312S (rs2293275) in the luteinizing hormone/human chorionic gonadotropin receptor (LHCGR) predicted cumulative live birth rate after in vitro fertilization (IVF). METHODS: A total of 665 women were consecutively enrolled for IVF during the period 2007-2016. Inclusion criteria were < 40 years of age, body mass index < 30 kg/m2, non-smoking, regular menstruation cycle of 21-35 days, and bilateral ovaries. A blood sample was drawn for endocrine hormonal analysis and for DNA extraction with subsequent genotyping of the FSHR N680S and LHCGR N312S polymorphisms. Statistical analyses were done on all completed IVF cycles. RESULTS: Women homozygous for S in both receptors combined (4S) had significantly higher live birth rate compared to those with other receptor variants when combining the first three IVF cycles (OR = 2.00, 95% CI [1.02, 3.92], p = 0.043). Cumulatively higher chance of live birth rate, during all IVF cycles, was also evident (HR = 1.89, 95% CI [1.00, 3.57], p = 0.049). CONCLUSIONS: Gonadotropin receptor variants are promising candidates for the prediction of the possibility to have a baby to take home after IVF treatment.

The impact of luteal serum progesterone levels on live birth rates-a prospective study of 602 IVF/ICSI cycles.

STUDY QUESTION: Is the chance of a live birth following IVF treatment and fresh embryo transfer affected by early and mid-luteal serum progesterone (P4) levels? SUMMARY ANSWER: Low as well as high serum P4 levels in the early and mid-luteal phase reduce the chance of a live birth following IVF treatment with fresh embryo transfer. WHAT IS KNOWN ALREADY: Data from non-human studies and studies of frozen-thawed embryo transfer cycles indicate that low as well as high P4 levels during the mid-luteal phase decrease the chance of pregnancy. The altered P4 pattern may disrupt the endometrial maturation leading to asynchrony between embryonic development and endometrial receptivity, thereby, compromising implantation and early development of pregnancy. STUDY DESIGN, SIZE, DURATION: Prospective multicenter cohort study of 602 women undergoing IVF treatment. Patients were recruited from four Danish public Fertility Centers from May 2014 to June 2017. The study population was unselected, thus, representing a normal everyday patient cohort. Patients were treated in a long GnRH-agonist protocol or a GnRH-antagonist protocol and triggered for final oocyte maturation with either hCG or a GnRH-agonist. The same vaginal luteal support regimen was applied in all patients. PARTICIPANTS/MATERIALS, SETTING, METHODS: Serum P4 levels from the early or mid-luteal phase were correlated to positive hCG and live birth rates (delivery > gestational week 20). Patients were divided into four P4 groups based on raw data of P4 serum levels and reproductive outcomes during early luteal phase (P4<60 nmol/l, P4 60-100 nmol/l, P4 101-400 nmol/l and P4>400 nmol/l) and during mid-luteal phase (P4<150 nmol/l, P4 150-250 nmol/l, P4 251-400 nmol/l and P4>400 nmol/l). MAIN RESULTS AND THE ROLE OF CHANCE: The optimal chance of pregnancy was achieved with serum P4 levels of 60-100 nmol/l in the early luteal phase whereas the optimal P4 level during the mid-luteal phase was 150-250 nmol/l. Below, but most distinctly above these levels, the chance of pregnancy was consistently reduced. With an early luteal P4 level of 60-100 nmol/l, the chance of a positive hCG-test was 73%, 95% CI: [59, 84] following cleavage stage embryo transfer. In contrast, with P4 levels >400 nmol/l, the chance of a positive hCG-test was significantly reduced to 35%, 95% CI: [17, 57], thus, an absolute risk difference of -38%, P = 0.01. A similar negative association between early luteal P4 and live birth rate was found, although it did not reach statistical significance. During the mid-luteal phase, a P4 level of 150-250 nmol/l resulted in an optimal chance of live birth: 54%, 95% CI: [37, 70] compared to 38%, 95% CI: [20, 60] with a P4 level >400 nmol/l, thus, an absolute risk difference of -16%, P = 0.14. All estimates were adjusted for maternal age, maternal BMI, study site, final follicle count and late follicular P4 levels. LIMITATIONS, REASONS FOR CAUTION: This study is the first to explore the possible upper and lower thresholds for luteal P4 following IVF treatment and fresh embryo transfer, and the optimal P4 ranges found in this study should be corroborated in future clinical trials. Furthermore, the P4 thresholds in this study only apply to fresh IVF cycles, using vaginal luteal phase support, as the optimal P4 level in cycles using intramuscular P4 may be different. WIDER IMPLICATIONS OF THE FINDINGS: Future studies are necessary to explore whether additional exogenous luteal P4 supplementation in the low P4 group could increase the chance of a live birth following fresh embryo transfer, and whether patients with luteal P4 levels >400 nmol/l would benefit from segmentation followed by subsequent transfer in frozen/thawed cycles. TRIAL REGISTRATION NUMBER: NCT02129998 (Clinicaltrials.gov). STUDY FUNDING/COMPETING INTEREST(S): L.H.T. received an unrestricted grant from Ferring Pharmaceuticals, Denmark, to support this study. P.H. received unrestricted research grants from MSD, Merck, Gedeon Richter and Ferring Pharmaceuticals outside of this work as well as honoraria for lectures from MSD, Merck and Gedeon Richter outside of this work. U.K. received honoraria for lectures from MSD and Ferring Pharmaceuticals outside of this work. C.A. received unrestricted research grants from MSD, IBSA, and Ferring Pharmaceuticals outside of this work as well as honoraria for lectures from MSD and IBSA. H.O.E. and B.B.P. received an unrestricted research grant from Gedeon Richter outside of this work. K.E., L.B., D.P. and B.H. have no conflict of interest. Furthermore, grants from 'The Health Research Fund of Central Denmark Region', 'The Research Foundation of the Hospital of Central Jutland', 'The Research Foundation of A.P. Møller', 'The Research Foundation of Aase & Ejnar Danielsen', 'The Research Foundation of Dagmar Marshall', 'The Research Foundation of Dir. Jacob Madsen & Hustru Olga Madsen', 'The Research Foundation of Fam. Hede Nielsen' and 'The Danish Medical Research Grant' supported conducting this study. The providers of funding were neither involved in the conduction of the study nor in the writing of the scientific report.

Environmental cadmium and lead exposure and anti-Müllerian hormone in pregnant women.

BACKGROUND: Anti-Müllerian Hormone (AMH) has been suggested as a marker for ovarian function. Cadmium and lead have been suggested to reduce female fecundity. In this study we aimed to investigate whether environmental exposure to cadmium and lead was associated with alterations in serum-AMH. MATERIALS AND METHOD: The associations between serum-AMH and whole blood cadmium or lead were investigated by general linear models in a population-based sample of 117 pregnant women. RESULTS: The mean concentrations of blood cadmium and lead were 0.71µg/L and 17.4µg/L, respectively. The mean serum-AMH was 17.3pmol/L. No association between lead and AMH was detected. In the cadmium analysis the adjusted mean AMH level (95% CI) in the highest exposure tertile was 12.4 (6.4;23.8) compared to 5.6 (2.7;11.4) in the lowest exposure tertile (p=0.06). CONCLUSION: The study provides suggestive evidence that environmental exposure to cadmium, but not lead, may alter the level of AMH.

Combined assessment of polymorphisms in the LHCGR and FSHR genes predict chance of pregnancy after in vitro fertilization.

STUDY QUESTION: Can gonadotrophin receptor variants separately or in combination, be used for the prediction of pregnancy chances in in vitro fertilization (IVF) trials? SUMMARY ANSWER: The luteinizing hormone/human chorionic gonadotrophin receptor (LHCGR) variant N312S and the follicle-stimulating hormone receptor (FSHR) variant N680S can be utilized for the prediction of pregnancy chances in women undergoing IVF. WHAT IS KNOWN ALREADY: The FSHR N680S polymorphism has been shown to affect the ovarian response in response to gonadotrophin treatment, while no information is currently available regarding variants of the LHCGR in this context. STUDY DESIGN, SIZE, DURATION: Cross-sectional study, duration from September 2010 to February 2015. Women undergoing IVF were consecutively enrolled and genetic variants compared between those who became pregnant and those who did not. The study was subsequently replicated in an independent sample. Granulosa cells from a subset of women were investigated regarding functionality of the genetic variants. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women undergoing IVF (n = 384) were enrolled in the study and genotyped. Clinical variables were retrieved from medical records. For replication, an additional group of n = 233 women was utilized. Granulosa cells from n = 135 women were isolated by flow cytometry, stimulated with Follitropin alpha or Menotropin, and the downstream targets 3',5'-cyclic adenosine monophosphate (cAMP) and inositol 1,4,5-trisphosphate (IP3) measured with enzyme-linked immunosorbent assay. MAIN RESULTS AND THE ROLE OF CHANCE: Women homozygous for serine (S) in both polymorphisms displayed higher pregnancy rates than women homozygous asparagine (N) (OR = 14.4, 95% CI: [1.65, 126], P = 0.016). Higher pregnancy rates were also evident for women carrying LHCGR S312, regardless of FSHR variant (OR = 1.61, 95% CI: [1.13, 2.29], P = 0.008). These women required higher doses of FSH for follicle recruitment than women homozygous N (161 versus 148 IU, P = 0.030). When combining the study cohort with the replication cohort (n = 606), even stronger associations with pregnancy rates were noted for the combined genotypes (OR = 11.5, 95% CI: [1.86, 71.0], P = 0.009) and for women carrying LHCGR S312 (OR = 1.49, 95% CI: [1.14, 1.96], P = 0.004). A linear significant trend with pregnancy rate and increasing number of G alleles was also evident in the merged study population (OR = 1.34, 95% CI: [1.10, 1.64], P = 0.004). A lower cAMP response in granulosa cells was noted following Follitropin alpha stimulation for women homozygous N in both polymorphisms, compared with women with other genotypes (0.901 pmol cAMP/mg total protein versus 2.19 pmol cAMP/mg total protein, P = 0.035). LIMITATIONS, REASONS FOR CAUTION: Due to racial differences in LHCGR genotype distribution, these results may not be applicable for all populations. WIDER IMPLICATIONS OF THE FINDINGS: Despite that >250 000 cycles of gonadotrophin stimulations are performed annually worldwide prior to IVF, it has not been possible to predict neither the pregnancy outcome, nor the response to the hormone with accuracy. If LHCGR and FSHR variants are recognized as biomarkers for chance of pregnancy, more individualized and thereby more efficient treatment modalities can be developed. STUDY FUNDING, COMPETING INTERESTS: This work was supported by Interreg IV A, EU (grant 167158) and ALF governments grant (F2014/354). Merck-Serono (Darmstadt, Germany) supported the enrollment of the subjects. The authors declare no conflict of interest.

Spermatozoa DNA damage measured by sperm chromatin structure assay (SCSA) and birth characteristics in children conceived by IVF and ICSI.

High levels of spermatozoa DNA damage hinder fertility in vivo but not in vitro. It is a source of worry that following in vitro fertilization (IVF) spermatozoa DNA damage, if not repaired by the oocyte, might have a negative impact on the offspring. The aim of this study was to assess if a high spermatozoa DNA Fragmentation Index (DFI) is associated with alterations in birthweight (BW) and/or gestational length in IVF children. One hundred and thirty-one singleton pregnancies established by standard IVF or intracytoplasmic sperm injection (ICSI) were included in the study. DFI was measured by sperm chromatin structure assay (SCSA) in semen samples used for fertilization. DFI was categorized as low and high, using 20, 30, 40 and 50% as cut-off levels. Birthweight, gestational age, as well as gestational age adjusted BW score were used in a linear regression model as end points For none of the tested birth characteristics, statistically significant differences between the groups with low and high DFI were seen regardless of whether 20, 30, 40 or 50% were used as cut-off levels, both when the IVF and ICSI data were merged or analysed separately. Spermatozoa DNA damage as assessed by SCSA is not associated with BW or gestational length in IVF and ICSI children.

A randomized controlled study comparing pain experience between a newly designed needle with a thin tip and a standard needle for oocyte aspiration.

UNLABELLED: BACKGROUND; Ultrasound-guided transvaginal oocyte retrieval is often performed under local anaesthesia on an outpatient basis. The objective of this study was to compare the overall pain experience of a newly designed reduced needle (RN) compared with a thicker standard needle (SN). METHODS: A prospective, randomized, multi-centre study was performed at four different clinics from June to December 2009. The oocyte aspiration was performed under local anaesthesia, either with a needle with a reduced diameter (0.9 mm) for the last 50 mm from the tip (RN) or with a SN (1.4 mm). A total of 257 patients were randomized (RN: n = 129; SN: n = 128). The primary endpoint was the overall pain experience self-assessed and registered by the patient on a visual analogue scale (VAS 0 mm = no pain to 100 mm = unbearable pain) immediately after the oocyte retrieval. Secondary end-points such as vaginal bleeding and several embryological parameters were also registered. RESULTS: The overall pain during the oocyte retrieval procedure was significantly lower in the RN group than in the SN group (mean 21.0 mm, SD 17.5 mm and median 19.0 mm versus mean 26.0 mm, SD 19.9 mm and median 24.0 mm; P = 0.040, difference between groups mean-5.0 mm, 95% CI: 9.7 to-0.4). This was also true when adjusting for baseline characteristics such as number of follicles, number of previous oocyte pick-up, body mass index and age, by a multiple linear regression analysis. Significantly more patients (40 of 126) had less than expected vaginal bleeding in the RN group when compared with the SN group (24 of 124; 32 versus 19%; P = 0.03 and 95% CI 1.7-23.0%). No differences were found between the two needles with regard to additional i.v. analgesia, aspiration time, oocyte recovery, fertilization, cleavage rate, number of good quality embryos, number of embryos for freezing and pregnancy rate. CONCLUSIONS: Oocyte aspiration performed with the newly designed thinner-tipped needle resulted in significantly less overall pain and less vaginal bleeding, without prolonging the retrieval procedure or influence the oocyte recovery rate, when compared with a SN. Clinicaltrials.gov: NCT00924885.

Sperm DNA integrity assessment in prediction of assisted reproduction technology outcome.

BACKGROUND: The sperm chromatin structure assay (SCSA) has been suggested as a predictor of fertility in vivo as well as in vitro. The available data however, have been based on limited numbers of treatments. We aimed to define the clinical role of SCSA in assisted reproduction. METHODS: A total of 998 cycles [387 intrauterine insemination (IUI), 388 IVF and 223 ICSI] from 637 couples were included. SCSA results were expressed as DNA fragmentation index (DFI) and high DNA stainable (HDS) cell fractions. Outcome parameters were biochemical pregnancy (BP), clinical pregnancy (CP) and delivery (D). RESULTS: For IUI, the odds ratios (ORs) for BP, CP and D were significantly lower for couples with DFI >30% as compared with those with DFI < or =30%. No statistical difference between the outcomes of ICSI versus IVF in the group with DFI < or =30% was seen. In the DFI >30% group, the results of ICSI were significantly better than those of IVF. CONCLUSIONS: DFI can be used as an independent predictor of fertility in couples undergoing IUI. As a result, we propose that all infertile men should be tested with SCSA as a supplement to the standard semen analysis. When DFI exceeds 30%, ICSI should be the method of choice.

Rescue of corpus luteum function with peri-ovulatory HCG supplementation in IVF/ICSI GnRH antagonist cycles in which ovulation was triggered with a GnRH agonist: a pilot study.

Previous studies found a poor clinical outcome when a GnRH agonist (GnRHa) was used to trigger ovulation in GnRH antagonist IVF/ICSI cycles. This study aimed to determine the clinical and endocrine effects as well the optimal timing of HCG supplementation. Forty-five normogonadotrophic IVF/ICSI patients following a flexible antagonist protocol were prospectively randomized (sealed envelopes) to triggering of ovulation with a single bolus of either 10,000 IU of HCG (group 1, n = 15) or 0.5 mg buserelin s.c. In addition, the GnRHa triggered group was randomized into two groups: group 2 (n = 17) was supplemented with HCG 1500 IU, 12 h after ovulation induction and group 3 (n = 13) was supplemented with HCG 1500 IU 35 h after ovulation induction. Group 1 and group 3 had significantly higher luteal phase concentrations of progesterone (P < 0.001) as compared with group 2. Moreover, the clinical pregnancy rate of groups 1 and 3 was similar and significantly higher (P < 0.02) than that of group 2. A larger study, however, is required to substantiate these differences. No differences were seen regarding mid-luteal inhibin A concentrations between the three groups. Triggering of ovulation with GnRHa supplemented with 1500 IU HCG 35 h later (group 3) seems to secure a normal luteal phase and a normal clinical pregnancy outcome.

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.

GnRH agonist (buserelin) or hCG for ovulation induction in GnRH antagonist IVF/ICSI cycles: a prospective randomized study.

BACKGROUND: We aimed to determine the efficacy of ovarian hyperstimulation protocols employing a GnRH antagonist to prevent a premature LH rise allowing final oocyte maturation and ovulation to be induced by a single bolus of either a GnRH agonist or hCG. METHODS: A total of 122 normogonadotrophic patients following a flexible antagonist protocol was stimulated with recombinant human FSH and prospectively randomized (sealed envelopes) to ovulation induction with a single bolus of either 0.5 mg buserelin s.c. (n = 55) or 10,000 IU of hCG (n = 67). A maximum of two embryos was transferred. Luteal support consisted of micronized progesterone vaginally, 90 mg a day, and estradiol, 4 mg a day per os. RESULTS: Ovulation was induced with GnRH agonist in 55 patients and hCG in 67 patients. Significantly more metaphase II (MII) oocytes were retrieved in the GnRH agonist group (P < 0.02). Significantly higher levels of LH and FSH (P < 0.001) and significantly lower levels of progesterone and estradiol (P < 0.001) were seen in the GnRH agonist group during the luteal phase. The implantation rate, 33/97 versus 3/89 (P < 0.001), clinical pregnancy rate, 36 versus 6% (P = 0.002), and rate of early pregnancy loss, 4% versus 79% (P = 0.005), were significantly in favour of hCG. CONCLUSIONS: Ovulation induction with a GnRH agonist resulted in significantly more MII oocytes. However, a significantly lower implantation rate and clinical pregnancy rate in addition to a significantly higher rate of early pregnancy loss was seen in the GnRH agonist group, most probably due to a luteal phase deficiency.

Reproductive outcome using a GnRH antagonist (cetrorelix) for luteolysis and follicular synchronization in poor responder IVF/ICSI patients treated with a flexible GnRH antagonist protocol.

To investigate the possible beneficial effect of a new stimulation protocol (termed 'CRASH') on the outcome of poor responder patients, a multicentre, prospective longitudinal study including a total of 36 women undergoing 72 IVF/intracytoplasmic sperm injection (ICSI) cycles with patients serving as their own controls, was conducted. A poor responder patient was defined as a patient with four or fewer oocytes extracted from five or fewer follicles and with a total FSH consumption exceeding 2000 IU in a preceding long agonist down-regulation protocol. The CRASH protocol included 3 mg of the gonadotrophin-releasing hormone (GnRH) antagonist cetrorelix given in the late luteal phase on cycle day 23. Stimulation with recombinant human FSH (rhFSH) started on cycle day 2, followed by a flexible GnRH antagonist protocol. The results showed significantly more follicles (5.4 versus 3.5), oocytes (4.3 versus 2.4) and transferable embryos (1.8 versus 0.8) with the CRASH protocol as compared with the preceding long protocol (P < 0.005 in all cases). The implantation rate and pregnancy rate per transfer was 18.4 and 38.5% respectively, approaching the clinical outcome of normal responder patients. The CRASH protocol thus may constitute an attractive alternative to conventional protocols for low responder patients, improving their clinical outcome.

Effects of recombinant LH supplementation in women undergoing assisted reproduction with GnRH agonist down-regulation and stimulation with recombinant FSH: an opening study.

Circulating endogenous concentrations of LH are reduced in women undergoing down-regulation with gonadotrophin-releasing hormone agonists (GnRHa) and ovarian stimulation with recombinant human FSH (r-hFSH). The effect of recombinant human LH (r-hLH) supplementation on ovarian response and pregnancy outcome was evaluated in a prospective randomized study (sealed envelopes) including 231 cycles. Normogonadotrophic women were stimulated with either r-hFSH or a combination of r-hFSH and r-hLH in a ratio of 2:1. LH supplementation was started from day 8 of the cycle. Blood samples for oestradiol, LH and androstendione were prospectively collected on days 1, 8 and on the day of aspiration and analysed retrospectively. Overall, the two groups did not differ with respect to pregnancy rate. In contrast, women aged > or =35 years responded to exogenous LH supplementation with significantly increased implantation rates and significantly reduced total FSH consumption as compared with the non-supplemented group. In addition, the implantation rate for a subgroup of patients with the highest endogenous LH concentrations (i.e. > or =1.99 IU/l) on day 8 was significantly increased by LH supplementation. Exogenous LH supplementation from day 8 has no detrimental effect on ovarian response and pregnancy outcome. On the contrary supplementation with r-hLH seems to benefit treatment outcome for women above 35 years of age and for the subgroup of women exhibiting LH concentrations above 1.99 IU/l on stimulation day 8.

A strategy for treatment of couples with unexplained infertility who failed to conceive after intrauterine insemination.

Couples with unexplained infertility treated unsuccessfully with intrauterine insemination often receive further treatment with IVF or intracytoplasmic sperm injection (ICSI). The aim of this study was to evaluate the frequency of fertilization and fertilization failure with respect to the method of fertilization used, when half of the sibling oocytes were fertilized by conventional IVF and insemination and the remainder by ICSI. Included was the first IVF/ICSI treatment of 248 unexplained infertile couples who had failed to conceive after three IUI cycles. An overall pregnancy rate per embryo transfer of 57% was observed. A significantly better fertilization rate was obtained after ICSI as compared with IVF (68 versus 46%) (P < 0.005), and total fertilization failure following ICSI and IVF treatment was seen in 4.4 and 25% of the cycles respectively. The group who experienced total fertilization failure after IVF had normal semen parameters, although significantly lower sperm concentration and motility as compared with the entire study group. Transfer of their ICSI-fertilized oocytes subsequently resulted in a pregnancy rate of 49.2% per transfer. The policy of splitting the sibling oocytes can effectively minimize complete fertilization failure while maintaining high chances of achieving a pregnancy. At the same time, the optimal fertilization method for subsequent treatment cycles can be determined.

The predictive value of sperm chromatin structure assay (SCSA) parameters for the outcome of intrauterine insemination, IVF and ICSI.

INTRODUCTION: Sperm chromatin integrity assessment has been suggested as a fertility predictor. The aim of this study was to examine the relationship between the results of sperm chromatin structure assay (SCSA) and the outcome of IVF, ICSI and intrauterine insemination (IUI). METHODS: A total of 306 consecutive couples undergoing assisted reproduction were included. IUI was performed in 131, IVF in 109 and ICSI in 66. SCSA results were expressed as DNA fractionation index (DFI) and highly DNA stainable (HDS) cell fractions. Reproductive outcome parameters were biochemical pregnancy (BP), clinical pregnancy (CP) and delivery (D). RESULTS: For IUI, the chance of pregnancy/delivery was significantly higher in the group with DFI 27% or HDS >10%. The odds ratios (ORs) (95% confidence intervals) were 20 (2.3-117), 16 (1.9-137) and 14 (1.6-110) for BP, CP and D, respectively. No statistical difference between the outcomes of IVF versus ICSI was observed in the group with DFI 27% group, however, the results of ICSI were significantly better than those of IVF. Comparing ICSI with IVF, the OR (95% CI) for BP was 26 (1.9-350). CONCLUSIONS: SCSA is a useful method for prediction of the outcome of assisted reproduction.

Day 3 versus day 5 embryo transfer: a prospective randomized study.

Transfer of embryos at the blastocyst stage has been associated with exceptionally high implantation rates. There are, however, only a few prospective randomized studies comparing day 3 versus day 5 embryo transfer. Furthermore, the number of embryos replaced in the day 3 group transfer is often higher than the number of blastocysts replaced, thereby affecting implantation rates. A total of 118 patients undergoing standard IVF/intracytoplasmic sperm injection who had developed at least three 8-cell embryos showing <20% extracellular fragmentation on day 3 were randomized for day 3 or day 5 transfer. A maximum of two embryos were replaced. In this prospective, randomized study the implantation and pregnancy potential of embryos transferred on day 3 or day 5 were compared. Equal numbers of embryos were replaced in the two groups. There was no statistically significant difference between day 3 and day 5 transfer regarding positive human chorionic gonadotrophin rates (70 versus 67%), clinical pregnancy rates (61 versus 51%), implantation rates (44 versus 37%), twinning rates (42 versus 41%) and rates of early pregnancy loss (15 versus 29%). Transfer of embryos on day 3 or 5 showed similar implantation rates when equal numbers of embryos were transferred. Embryo transfer at the blastocyst stage seems to have no advantage over day 3 transfer in patients with more than two 8-cell embryos showing less than 20% fragmentation on day 3.

Ovarian response and pregnancy outcome related to mid-follicular LH levels in women undergoing assisted reproduction with GnRH agonist down-regulation and recombinant FSH stimulation.

BACKGROUND: The effect of LH levels on stimulation day 8 on ovarian response and pregnancy outcome were evaluated in women receiving pituitary down-regulation with GnRH agonists (0.8 mg Suprefact) s.c. daily until pituitary down-regulation and 0.4 mg/day during ovarian stimulation) and ovarian stimulation with recombinant FSH. METHODS: Blood samples were prospectively collected from a total of 207 normal women undergoing assisted reproduction and analysed retrospectively. Based on LH levels on stimulation day 8 patients were divided into four groups: <0.5, 0.51-1.0, 1.01-1.5, >1.51 IU/l. RESULTS: Estradiol levels on day 8 and estradiol per oocyte retrieved showed a highly significant correlation with LH concentrations on day 8. The total consumption of exogenous FSH and duration of gonadotrophin stimulation was inversely related to LH levels on day 8 (P < 0.002). The frequencies of fertilization and clinical pregnancies were superior in the two middle groups. Only 12% of the patients showed LH levels <0.5 IU/l, which, however, may be explained by the particular mode and doses of GnRH agonist used. CONCLUSIONS: Circulating levels of LH on day 8 have a significant impact on ovarian response and pregnancy outcome. LH should neither be too high nor too low. The mode of administration and the dose of the GnRH agonist used may determine the number of women who experience LH levels below 0.5 IU/l.

Recombinant human albumin as protein source in culture media used for IVF: a prospective randomized study.

Oocytes obtained from 85 women undergoing IVF/intracytoplasmic sperm injection (ICSI) treatment were randomly selected for culture in media containing either human serum albumin (HSA), (n = 42) or recombinant human albumin (rHA), (n = 43). At the time of transfer, the two embryos with the overall best morphology were selected on day 2, 3 or 5. Comparable rates of fertilization, cleavage, blastocyst formation and implantation were observed in both groups. The frequency of pregnancy and early pregnancy loss were also equal. It is concluded that culture media containing rHA seem to produce embryos of high quality comparable to media containing HSA. Therefore, rHA may replace HSA as a protein source in culture media for IVF and may reduce risks of prion contamination and transmission of plasma derived impurities.