A distinctive epigenetic ageing profile in human granulosa cells.

STUDY QUESTION: Does women's age affect the DNA methylation (DNAm) profile differently in mural granulosa cells (MGCs) from other somatic cells? SUMMARY ANSWER: Accumulation of epimutations by age and a higher number of age-related differentially methylated regions (DMR) in MGCs were found compared to leukocytes from the same woman, suggesting that the MGCs have a distinctive epigenetic profile. WHAT IS KNOWN ALREADY: The mechanisms underlying the decline in women's fertility from the mid-30s remain to be fully elucidated. The DNAm age of many healthy tissues changes predictably with and follows chronological age, but DNAm age in some reproductive tissues has been shown to depart from chronological age (older: endometrium; younger: cumulus cells, spermatozoa). STUDY DESIGN, SIZE, DURATION: This study is a multicenter cohort study based on retrospective analysis of prospectively collected data and material derived from healthy women undergoing IVF or ICSI treatment following ovarian stimulation with antagonist protocol. One hundred and nineteen women were included from September 2016 to June 2018 from four clinics in Denmark and Sweden. PARTICIPANTS/MATERIALS, SETTING, METHODS: Blood samples were obtained from 118 healthy women with varying ovarian reserve status. MGCs were collected from 63 of the 119 women by isolation from pooled follicles immediately after oocyte retrieval. DNA from leukocytes and MGCs was extracted and analysed with a genome-wide methylation array. Data from the methylation array were processed using the ENmix package. Subsequently, DNAm age was calculated using established and tailored age predictors and DMRs were analysed with the DMRcate package. MAIN RESULTS AND ROLE OF CHANCE: Using established age predictors, DNAm age in MGCs was found to be considerable younger and constant (average: 2.7 years) compared to chronological age (average: 33.9 years). A Granulosa Cell clock able to predict the age of both MGCs (average: 32.4 years) and leukocytes (average: 38.8 years) was successfully developed. MGCs differed from leukocytes in having a higher number of epimutations (P = 0.003) but predicted telomere lengths unaffected by age (Pearson's correlation coefficient = -0.1, P = 0.47). DMRs associated with age (age-DMRs) were identified in MGCs (n = 335) and in leukocytes (n = 1) with a significant enrichment in MGCs for genes involved in RNA processing (45 genes, P = 3.96 × 10-08) and gene expression (152 genes, P = 2.3 × 10-06). The top age-DMRs included the metastable epiallele VTRNA2-1, the DNAm regulator ZFP57 and the anti-Müllerian hormone (AMH) gene. The apparent discordance between different epigenetic measures of age in MGCs suggests that they reflect difference stages in the MGC life cycle. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: No gene expression data were available to associate with the epigenetic findings. The MGCs are collected during ovarian stimulation, which may influence DNAm; however, no correlation between FSH dose and number of epimutations was found. WIDER IMPLICATIONS OF THE FINDINGS: Our findings underline that the somatic compartment of the follicle follows a different methylation trajectory with age than other somatic cells. The higher number of epimutations and age-DMRs in MGCs suggest that their function is affected by age. STUDY FUNDING/COMPETING INTEREST(S): This project is part of ReproUnion collaborative study, co-financed by the European Union, Interreg V ÖKS, the Danish National Research Foundation and the European Research Council. The authors declare no conflict of interest.

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.

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.

Sperm chromatin structure assay in prediction of in vitro fertilization outcome.

Sperm DNA fragmentation index (DFI) assessed by sperm chromatin structure assay is a valuable tool for prediction of fertility in vivo. Previous studies on DFI as predictor of in vitro fertilization (IVF) outcome, based on relatively small materials, gave contradictory results. The present study examines, in a large cohort, the association between sperm DFI and the outcome of IVF/ICSI procedure. The study is based on 1633 IVF or ICSI cycles performed at the Reproductive Medicine Centre, Skåne University Hospital, Malmö, Sweden, between May 2007 and March 2013. DFI values were categorized into four intervals: DFI ≤ 10% (reference group), 10% < DFI ≤ 20%, 20% < DFI ≤ 30%, DFI > 30%. For the three latter intervals, the following outcomes of IVF/ICSI procedures were analyzed in relation to the reference group: fertilization, good quality embryo, pregnancy, miscarriage, and live births. In the standard IVF group, a significant negative association between DFI and fertilization rate was found. When calculated per ovum pick-up (OPU) Odds Ratios (ORs) for at least one good quality embryo (GQE) were significantly lower in the standard IVF group if DFI > 20%. OR for live birth calculated per OPU was significantly lower in standard IVF group if DFI > 20% (OR 0.61; 95% CI: 0.38-0.97; p = 0.04). No such associations were seen in the ICSI group. OR for live birth by ICSI compared to IVF were statistically significantly higher for DFI > 20% (OR 1.7; 95% CI: 1.0-2.9; p = 0.05). OR for miscarriage was significantly increased for DFI > 40% (OR 3.8; 95% CI: 1.2-12; p = 0.02). The results suggest that ICSI might be a preferred method of in vitro treatment in cases with high DFI. Efforts should be made to find options for pharmacologically induced reduction of DFI. The study was based on retrospectively collected data and prospective studies confirming the superiority of ICSI in cases with high DFI are warranted.

No association between body mass index and sperm DNA integrity.

STUDY QUESTION: Is overweight associated with impaired sperm DNA integrity? SUMMARY ANSWER: High body mass index (BMI) is not associated with impaired sperm DNA integrity as assessed by the DNA Fragmentation Index (DFI). WHAT IS KNOWN ALREADY: Previous studies, based on fewer subjects and including mainly subfertile men, have shown conflicting results regarding the influence of overweight and obesity on sperm DNA integrity. STUDY DESIGN, SIZE, DURATION: This cross-sectional study was based on semen samples from 1503 men from the general population. PARTICIPANTS/MATERIALS, SETTING, METHODS: We included two cohorts (cohort A and B) of military recruits (n = 275, n = 304, respectively), one group (cohort C) of fertile men and men without known fertility problems (n = 724), and one group (cohort D) of men between 19 and 40 years without known fertility problems (n = 200). In all cohorts, data were available on BMI, DFI as measured by the sperm chromatin structure assay (SCSA), standard semen characteristics, and potential confounders (age, abstinence time, smoking habits). The subjects were categorized according to BMI into four groups: underweight (<18.5 kg/m(2)), normal weight (18.5-24.9 kg/m(2)), overweight (25.0-29.9 kg/m(2)) and obese (≥30.0 kg/m(2)). Using a linear regression model, the inter-group differences in DFI were calculated. Furthermore with the normal-weight group as the reference, the odds ratios (ORs) for DFI > 20% and DFI > 30%, were calculated for the other groups. Calculations were made for the material as a whole and after exclusion of cohort C which included proven fertile men. MAIN RESULTS AND THE ROLE OF CHANCE: We found that normal-weight men had significantly higher DFI than overweight men, with a mean difference of 1.13% (95% CI: 1.05-1.22%); P = 0.001). Overweight men had a reduced risk of having DFI ≥ 20% and DFI ≥ 30%, compared with normal-weight men; adjusted odds ratio (OR) = 0.61 (95% CI: 0.42-0.88; P < 0.01) and adjusted OR = 0.48 (95% CI: 0.28-0.84; P < 0.01), respectively. When excluding cohort C, the statistical significance was lost. Regarding standard semen parameters, we found that obese men had a higher percentage of progressive motile spermatozoa than normal-weight men; mean difference 1.15% (95% CI: 1.02-1.30%, P < 0.05) but the significance was lost when excluding cohort C. All other standard semen parameters were unaffected by BMI. LIMITATIONS, REASONS FOR CAUTION: A main limitation might be the cross-sectional nature of the data. Furthermore our study included a significant proportion of men with proven fertility (75% of cohort C, n = 550), and could therefore be biased toward fertility. WIDER IMPLICATIONS OF THE FINDINGS: Our study indicates that overweight per se is not associated with a higher level of sperm DNA damage. STUDY FUNDING/COMPETING INTERESTS: This research has been given grants from the following: EU 5th and 7th framework program (Inuendo and Clear projects, [Contracts no. QLK4-CT-2001-00202 and FP7-ENV-2008-1-226217)]), the Swedish Research Council (Grants No. 2007-2590, 521-2004-6072 and 521-2002-3907); the Swedish Governmental Funding for Clinical Research, Skåne county council's research and development foundation, MAS Funds, University Hospital MAS Foundation in Malmö, Crafoordska Fund, Ove Tulefjords Fund, Foundation for Urological Research, Fundacion Federico SA, and Gunnar Nilssons Cancer Fund. The authors declare that there are no conflicts of interest.

Prevalence of high DNA fragmentation index in male partners of unexplained infertile couples.

The sperm chromatin structure assay (SCSA) parameter DNA fragmentation Index (DFI) is a valuable tool for prediction of fertility in vivo. Clinical data show that a DFI above 30% is associated with very low chance for achieving pregnancy by natural conception or by insemination. Already when DFI is above 20% the chance of natural pregnancy is reduced, this despite normal conventional semen parameters. The aim of the present study was to investigate the prevalence of high DFI in male partners of unexplained infertile couples to further identification of male factors contributing to subfertility. Among 212 consecutive men under infertility investigation, 122 cases with the diagnosis 'unexplained infertility' were identified. For all but three, SCSA data were available. The percentage of couples with diagnosis 'unexplained infertility' in which the male partner has DFI >20% or DFI >30% was calculated. In the group diagnosed with 'unexplained infertility' 17.7% of the men (95% CI 10.8-24.5) presented with 20 ≤DFI <30 and 8.4% (95% CI 3.40-13.4) had DFI ≥30%. A significant part of men diagnosed as unexplained infertile according to traditional diagnostic methods has remarkably high degrees of fragmented sperm DNA. Apart from adding to our understanding of biology of infertility our finding has clinical implications. Couples in which the DFI of the male partner is high can avoid prolonged attempts to become spontaneously pregnant or referral for intrauterine insemination, both having low chances of leading to conception.

No increased sperm DNA fragmentation index in semen containing human papillomavirus or herpesvirus.

It remains unknown whether human papillomaviruses (HPVs) or human herpesviruses (HHVs) in semen affect sperm DNA integrity. We investigated whether the presence of these viruses in semen was associated with an elevated sperm DNA fragmentation index. Semen from 76 sperm donors was examined by a PCR-based hybridization array that identifies all HHVs and 35 of the most common HPVs. Sperm DNA integrity was determined by the sperm chromatin structure assay. HPVs or HHVs, or both, were found in 57% of semen samples; however, sperm DNA fragmentation index was not increased in semen containing these viruses.

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.

Intra-individual variation of the sperm chromatin structure assay DNA fragmentation index in men from infertile couples.

BACKGROUND: The sperm chromatin structure assay (SCSA) is a valuable tool for prediction of fertility in vivo, with DNA fragmentation index (DFI) of 30% as a clinically useful cut-off level. Previous studies on fertile men have shown a high level of repeatability, with an intra-individual variability in DFI of ≈ 10%. However, conflicting data on how much the DFI fluctuates within individuals exist. The aim of the present study was to investigate the intra-individual variation of DFI in order to further evaluate the clinical use of SCSA. METHODS: Among 2409 consecutive men under infertility investigation, repeated SCSA analyses were performed on 616 samples from men between 18 and 66 years of age. The coefficient of variation (CV) for DFI was calculated. For each patient, we also analyzed whether the DFI value in tests I and II switched the category from <30 to >30%, or vice versa. RESULTS: Mean CV for DFI for men with at least two SCSA analyses within a 30-month period was 30.1% (SD 21.5). Compared with the first test, 85% (95% confidence interval: 82-87%) of the men remained on the same side of the cut-off point of 30%. CONCLUSIONS: Despite showing a high intra-individual CV for DFI, 85% of the men from infertile couples did not change category between tests, with respect to the cut-off level of 30%. Thus, using the previously established DFI cut-off value of 30%, a single SCSA analysis has a high predictive value for assessing fertility in vivo.

Evaluation of male fertility potential by Toluidine Blue test for sperm chromatin structure assessment.

BACKGROUND: We have previously suggested that the Toluidine Blue (TB) test can be used for sperm chromatin structure assessment. In this study, we wished to evaluate the clinical applicability of the TB test in assessing male fertility potential using well-defined groups of fertile and infertile men. METHODS: Sixty-three fertile and 79 infertile men were tested. Infertility thresholds for the proportion of sperm with abnormal [TB dark cells (TBDCs)] and normal [TB light cells (TBLCs)] chromatin structure were set by the ROC curve analysis. RESULTS: Thresholds of 45% TBDC and 20% TBLC were highly predictive for infertility (specificity of the test: 92 and 90%, respectively), but they were poor predictors of the fertility (sensitivity of the test: 42 and 32%, respectively). Odds ratio for infertility was 7.5 [95% confidence interval (CI): 2.7-20.8] when the 45% TBDC threshold was used and 4.4 (95% CI: 1.7-11.6) when the 20% TBLC threshold was used. CONCLUSIONS: The TB test can be suggested for clinical use as a complementary test for standard semen analysis to diagnose male infertility.

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.

Intra-individual variation in sperm chromatin structure assay parameters in men from infertile couples: clinical implications.

BACKGROUND: Sperm DNA integrity is an important factor in the prognosis of male fertility. In this study, we investigated intra-individual variation of sperm chromatin structure assay (SCSA) parameters in infertility patients undergoing assisted reproductive techniques (ARTs). METHODS: Retrospective study of 282 consecutive patients referred for ART [intrauterine insemination (IUI), IVF or ICSI] with repeated (between 2 and 5) SCSA measurements. RESULTS: Mean coefficient of variation (CV) of DNA Fragmentation Index (DFI) for repeated SCSA measurements was 29%. A high proportion [37%; 95% confidence interval (CI): 27%, 49%] of patients with DFI >30% in the first test had DFI <30% in the second test. Also, a considerable proportion (27%; 95% CI : 16%, 40%) of patients with 21-30% DFI values in the first test had DFI >30% in the second test. CONCLUSIONS: Intra-individual variability in DFI is significant, therefore repeated SCSA measurements are recommended. The biological mechanisms behind these variations remain to be elucidated.

Sperm chromatin structure and male fertility: biological and clinical aspects.

Sperm chromatin/DNA integrity is essential for the accurate transmission of paternal genetic information, and normal sperm chromatin structure is important for sperm fertilizing ability. The routine examination of semen, which includes sperm concentration, motility and morphology, does not identify defects in sperm chromatin structure. The origin of sperm DNA damage and a variety of methods for its assessment are described. Evaluation of sperm DNA damage appears to be a useful tool for assessing male fertility potential both in vivo and in vitro. The possible impact of sperm DNA defects on the offspring is also discussed.

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.