Paul Serhal

Antral follicle count, anti-mullerian hormone and inhibin B: predictors of ovarian response in assisted reproductive technology?

Objective  The objective of this study was to evaluate the relationship between anti‐mullerian hormone (AMH), inhibin B and antral follicle count (AFC) with ovarian response.

Unexplained infertility—the value of Pergonal * superovulation combined with intrauterine insemination

Sixty-two women with unexplained infertility were studied. Fifteen (group 1) had timed intrauterine insemination (IUI), 25 (group 2) were treated by Pergonal (Serono Laboratories, Ltd., Welwyn Garden City, England) superovulation, and 22 (group 3) underwent Pergonal superovulation combined with IUI. Where Pergonal treatment was followed by insemination, a significantly greater pregnancy rate per cycle (P less than 0.05) was achieved, whether this group of patients was compared with those treated by IUI alone or with those treated with Pergonal alone. Moreover, the pregnancy rate in group 3 was comparable to that reported following gamete intrafallopian transfer (GIFT). The authors therefore suggest this form of treatment for patients with unexplained infertility prior to their referral to the more invasive procedure of GIFT.

FISH analysis on day 5 post-insemination of human arrested and blastocyst stage embryos.

Preimplantation genetic diagnosis (PGD) is usually performed on cleavage stage embryos on day 3 post‐insemination. Fluorescent in situ hybridization (FISH) has revealed four groups of chromosome patterns in embryos at this stage: uniformly normal, uniformly abnormal, mosaic and chaotic. Recently, some in vitro fertilization (IVF) clinics have started to perform blastocyst stage transfer. In blastocysts, conventional karyotyping has shown that all four groups of chromosome patterns are observed. In the present study, embryos were cultured to day 5 and were subject to a two‐round multicolour FISH procedure for chromosome analysis to ensure almost every nucleus was examined. Probes for chromosomes X, Y and 18 were used in the first round and those for chromosomes 13 and 21 in the second round. Twenty arrested embryos (274 cells) and 19 blastocyst stage embryos (1272 cells) were analysed. Four arrested embryos and two blastocysts were uniformly diploid. The remaining 33 embryos were mosaic, including 17 blastocysts. Most of the blastocysts had a high proportion of diploid cells while in the arrested embryos, this proportion varied widely. For PGD, this high prevalence of mosaicism persisting to the blastocyst stage may pose problems similar to mosaicism in cleavage stage embryos. Copyright

ANALYSIS OF 1071 GIFT PROCEDURES—THE CASE FOR A FLEXIBLE APPROACH TO TREATMENT

An analysis of the outcome of first gamete intrafallopian transfers for 1071 women indicates that for those aged 40 years or more all the oocytes had to be transferred to obtain a 19.2% pregnancy rate. In this age-group pregnancy rate and multiple pregnancy rate were significantly lower than those for younger women. Success rate, but not multiple pregnancy rate, was significantly higher in the group of women from whom 11 or more oocytes were recovered and transferred after ovulation induction than when only 1-4 oocytes were recovered and transferred. The findings suggest that the number of oocytes transferred should depend on clinical circumstances.

Fall in implantation rates following ICSI with sperm with high DNA fragmentation

BACKGROUND There is considerable uncertainty as to the significance of a high sperm DNA fragmentation index (DFI) for achieving a successful pregnancy. METHODS The sperm DFI of 124 patients undergoing 192 IVF cycles and of 96 patients undergoing 155 ICSI cycles was determined using the sperm chromatin structure assay on neat sperm. RESULTS The rate of continuing pregnancies in ICSI cycles (but not in IVF cycles) showed significant negative correlation (r = -0.184, P = 0.022) with the DFI value. A threshold value of DFI which showed a significant difference (P = 0.005) in rate of continuing pregnancies between higher and lower DFI levels was found for ICSI cycles to be > or = 19%, but no such threshold was found for IVF cycles. However, if the threshold of > or = 30% was used for IVF cycles there was a non-significant lowering of the rates of continuing pregnancy and implantation at the higher DFI levels. DFI level had no effect on fertilization rate or on the percentage of embryos having more than 4 cells at Day 3 after fertilization. A high DFI level had a marked significant effect (P = 0.001) on implantation rate in ICSI cycles but not in IVF cycles. A significant positive correlation (r = 0.268, P = 0.001) between DFI and sperm midpiece defects was also noted in the ICSI patients. CONCLUSIONS These observations may help to resolve the issues about how, and to what extent, sperm DNA damage impacts upon the success of IVF and ICSI procedures.

Expression profiling of DNA repair genes in human oocytes and blastocysts using microarrays

BACKGROUND The early preimplantation embryo relies on mRNA and protein from the oocyte to detect DNA damage and activate DNA repair, cell cycle arrest or apoptosis. Expression of some repair genes has been detected in mammalian oocytes and embryos; however, little is known about DNA repair gene expression in human blastocysts. In this study, DNA repair gene expression was investigated in human oocytes and blastocysts to identify the pathways involved at these stages and detect potential differences in repair mechanisms pre- and post-embryonic genome activation. METHODS Triplicate sets of pooled metaphase II oocytes or blastocysts were processed for analysis using the Human Genome Survey Microarrays V2.0 (Applied Biosystems). RESULTS Of 154 DNA repair genes investigated, 109 were detected in blastocysts and 107 in oocytes. Among differentially expressed DNA repair genes, 40/55 (73%) had lower expression levels in blastocysts compared with oocytes (P < 0.05, fold change >3). CONCLUSION Despite experimental limitations due to culture or freezing and thawing of samples, large numbers of repair genes were detected indicating that all DNA repair pathways are potentially functional in human oocytes and blastocysts. The higher mRNA level for most repair genes in oocytes compared with blastocysts ensures sufficient availability of template until embryonic genome activation.

Ovum donation—a simplified approach

To date, ovum donation (OD) has involved luteinizing hormone (LH) synchronization between recipient and donor for normally cycling women, and a complex steroid replacement regimen given on a sequential and incremental basis for women with primary or secondary ovarian failure. The authors designed a simple hormonal regimen applicable to both normally cycling women starting early in the cycle, and to those with ovarian failure. It consists of administering 2 mg estradiol (E2) valerate orally three or four times daily, augmented with either 100 mg progesterone (P) in ethyl oleate intramuscularly daily or 100 mg oral progesterone (P) orally three times daily, starting on the day preceding the recovery of the donated oocytes. Gamete intrafallopian transfer procedure was undertaken for women with patent tubes and in vitro fertilization for those with obstructed tubes. The authors report their preliminary experience with 17 women who underwent ovum donation.

Simultaneous evaluation of basal follicle-stimulating hormone and 17β-estradiol response to gonadotropin-releasing hormone analogue stimulation: an improved predictor of ovarian reserve

OBJECTIVE To compare different predictors of ovarian reserve. DESIGN Prospective study. SETTING The Assisted Conception Unit, University College London Hospitals. PATIENT(S) One hundred seventy-seven patients undergoing IVF treatment. INTERVENTION(S) Blood samples were collected on cycle day 2 to determine basal levels of FSH and 17beta-E2 and the FSH/LH ratio, and on cycle days 3 and 4 to assess the increase in FSH (deltaFSH) and 17beta-E2 (deltaE2) after the commencement of GnRH analogue (GnRH-a) stimulation. Ultrasound scans were performed during ovarian stimulation to assess the number of follicles. MAIN OUTCOME MEASURE(S) Day 2 FSH and 17beta-E2 levels, the FSH/LH ratio, and the deltaFSH and deltaE2 after the commencement of GnRH-a stimulation were correlated with the number of follicles obtained after ovarian stimulation. RESULT(S) All the possible predictors considered, except for the day 2 E2 level and the deltaFSH, correlated significantly with the ovarian response. The best single correlation was between the number of follicles and the deltaE2 (GnRH-a test). When the FSH level was evaluated simultaneously, the correlation was strengthened, resulting in a better negative predictive value. CONCLUSION(S) Simultaneous evaluation of basal levels of FSH and of the response of E2 to GnRH-a stimulation seems to be the best marker of ovarian reserve and a sensitive predictor of response to ovarian stimulation in patients undergoing IVF treatment.

Relationship between male reproductive hormones, sperm DNA damage and markers of oxidative stress in infertility

This study investigated the relationship between male reproductive hormones and sperm DNA damage and markers of oxidative stress in men undergoing infertility evaluation for male factor (n = 66) and non-male factor (n = 63) infertility. Semen samples were analysed for DNA fragmentation index (DFI). Serum samples were analysed for FSH, inhibin B, anti-Müllerian hormone (AMH), testosterone and total antioxidant capacity (TAC). Serum inhibin B was significantly lower in the male factor group compared with the non-male factor group. Inhibin B showed a positive correlation with sperm concentration and motility, and serum AMH showed a positive correlation with sperm concentration and semen volume. DFI was 3-fold higher in the male factor group and showed a negative correlation with sperm motility. Blood plasma TAC was negatively related to sperm concentration. The results confirm that AMH and inhibin B are markers of Sertoli cell function. Sperm DNA damage is moderately increased in male factor infertility, and is negatively associated with sperm motility. A negative association between antioxidant activity and sperm concentration suggests that even minimal oxidative stress may influence sperm concentration. However, there was no significant relationship between hormone concentrations, sperm DNA damage and total antioxidant capacity, suggesting other mechanisms for sperm dysfunction.

A successful strategy for preimplantation genetic diagnosis of myotonic dystrophy using multiplex fluorescent PCR

The most common form of inherited muscular dystrophy in adults is myotonic dystrophy (DM), an autosomal‐dominant disease caused by the expansion of an unstable CTG repeat sequence in the 3′ untranslated region of the myotonin protein kinase (DMPK) gene. Expanded (mutant) CTG repeat sequences are refractory to conventional PCR, but alleles with a number of repeats within the normal range can be readily amplified and detected. Preimplantation genetic diagnosis (PGD) of DM has been successfully applied. However, a misdiagnosis using the reported protocol was recently documented. Two new PGD protocols for DM have been developed which utilise multiplex fluorescent PCR. Ideally a linked polymorphic marker, APOC2, is amplified in addition to the normal DMPK alleles, thus providing a back‐up diagnostic result. However, the two couples reported in the present study were not fully informative at the APOC2 locus and so an unlinked short tandem repeat (STR) marker, D21S1414, was substituted. The highly polymorphic nature of the D21S1414, DMPK and APOC2 loci means that a very simple genetic fingerprint can be generated by analyses of these loci. This allows most DNA contaminants to be detected. Contamination is a significant problem for PGD and is the primary reason for the inclusion of D21S1414 and APOC2 in this protocol. This paper reports the first clinical experience and pregnancies following PGD for DM using a multiplex fluorescent PCR protocol. Copyright