Bulent Urman

Maternal methylation imprints on human chromosome 15 are established during or after fertilization.

Prader-Willi syndrome (PWS) is a neurogenetic disorder that results from the lack of transcripts expressed from the paternal copy of the imprinted chromosomal region 15q11–q13 (refs. 1,2). In some patients, this is associated with a deletion of the SNURF-SNRPN exon 1 region inherited from the paternal grandmother and the presence of a maternal imprint on the paternal chromosome. Assuming that imprints are reset in the germ line, we and others have suggested that this region constitutes part of the 15q imprinting center (IC) and is important for the maternal to paternal imprint switch in the male germ line. Here we report that sperm DNA from two males with an IC deletion had a normal paternal methylation pattern along 15q11–q13. Similar findings were made in a mouse model. Our results indicate that the incorrect maternal methylation imprint in IC deletion patients is established de novo after fertilization. Moreover, we found that CpG-rich regions in SNURF-SNRPN and NDN, which in somatic tissues are methylated on the maternal allele, are hypomethylated in unfertilized human oocytes. Our results indicate that the normal maternal methylation imprints in 15q11–q13 also are established during or after fertilization.

A randomized controlled study of human Day 3 embryo cryopreservation by slow freezing or vitrification: vitrification is associated with higher survival, metabolism and blastocyst formation†

BACKGROUND The aim of this study was to compare two methods of cryopreservation for the cleavage-stage human embryo: slow freezing and vitrification. METHODS A total of 466 Day 3 embryos, donated with consent, underwent cryopreservation by either slow freezing in straws or vitrification using the cryoloop. The vitrification procedure did not include dimethyl sulfoxide, but rather employed ethylene glycol and 1,2-propanediol as the cryoprotectants. Survival, embryonic metabolism and subsequent development to the blastocyst were used to determine the efficacy of the two procedures. RESULTS Significantly, more embryos survived the vitrification procedure (222/234, 94.8%) than slow freezing (206/232, 88.7%; P < 0.05). Consistent with this observation, pyruvate uptake was significantly greater in the vitrification group, reflecting a higher metabolic rate. Development to the blastocyst was also higher following vitrification (134/222, 60.3%) than following freezing (106/206, 49.5%; P < 0.05). In a separate cohort of 73 patients who had their supernumerary embryos cyropreserved with vitrification, the resulting implantation rate and clinical pregnancy rate were 30 and 49%, respectively. CONCLUSIONS Analysis of metabolism revealed that vitrification had less impact on the metabolic rate of the embryo than freezing, which was reflected in higher survival rate and subsequent development in vitro. Excellent pregnancy outcomes followed the warming and transfer of vitrified cleavage-stage embryos. These data provide further evidence that vitrification imparts less trauma to cells and is, therefore, a more effective means of cryopreserving the human embryo than conventional slow freezing. Clinicaltrials.gov identifier: NCT00608010.

Understanding follicle growth in vivo

Ovarian reserve is determined by the number of primordial follicles in the ovary. Quiescent primordial follicles are activated for growth and pass through stages of development before they reach the antral stage. Then a cohort of antral follicles is recruited for further growth, dominance and ovulation under the cyclic stimulation of gonadotrophins. What triggers the initiation of growth in primordial follicles has remained a mystery for decades. However, recent studies on mutant mouse models have shown that primordial follicles are maintained in a dormant state by the actions of various inhibitory molecules to preserve the follicle pool, such as the transcription factor Foxo3a, PTEN (phosphotase and tensin homolog deleted on chromosome 10) and Tsc-1 (tumour suppressor tuberous schlerosis complex). Mice with deletions of these oocyte-specific genes exhibit premature activation of dormant primordial follicles, and all primordial follicles become depleted in early adulthood, causing premature ovarian failure. Other oocyte and somatic cell-derived growth factors are also involved in the early, gonadotrophin-independent phase of follicle growth via autocrine and paracrine interactions. Interestingly, some of these factors also play critical roles at later stages of follicle growth, such as the process of selecting the dominant follicle, by modifying the response of the follicles to gonadotrophins and inhibiting premature luteinization. Therefore, a thorough understanding of the molecular aspects of folliculogenesis is of paramount importance in the context of translational medicine and future clinical applications in human reproduction.

Effect of oocyte morphology on embryo development and implantation

Assessment of oocyte morphology is a difficult task, since underlying mechanisms that change the appearance of the oocyte are multifactorial and complex. Significant morphological variations are known to exist among oocytes that may affect the developmental competence and implantation potential of the derived embryo. Morphological variations of the oocyte may result from intrinsic factors such as age and genetic defects or extrinsic factors such as stimulation protocols, culture conditions, and nutrition. The effect of these morphological variations of the oocyte on embryo development and implantation, however, is not conclusively defined because of methodological flaws inherent to most of the studies in the literature. This review will mainly discuss morphological markers of oocyte quality/viability in relation to the oocyte morphology and attempt to clarify whether morphological evaluation of the oocyte can be utilized for predicting the implantation potential of the derived embryo.

Blastocyst quality affects the success of blastocyst-stage embryo transfer

OBJECTIVE To determine the relationship between blastocyst quality and the results of embryo transfer at the blastocyst stage. DESIGN Retrospective case analysis. SETTING Tertiary care private hospital IVF center. PATIENT(S) A total of 350 blastocyst-stage embryo transfer cycles. INTERVENTION(S) In vitro culture to the blastocyst stage was undertaken in 350 ICSI cycles where four or more cleavage-stage embryos were available on day 3. MAIN OUTCOME MEASURE(S) Relationship between blastocyst quality and implantation and clinical and multiple pregnancy rates. RESULT(S) Transfer of at least one grade 1 or grade 2 blastocyst or one hatching blastocyst was associated with very high implantation and pregnancy rates. However, transfer of grade 3 blastocysts yielded very low implantation and pregnancy rates. CONCLUSION(S) There appears to be a strong correlation between blastocyst quality and success of blastocyst transfer.

Clinical outcome of intracytoplasmic injection of spermatozoa morphologically selected under high magnification: a prospective randomized study

Recent evidence shows that the selection of spermatozoa based on the analysis of morphology under high magnification (×6000) may have a positive impact on embryo development in cases with severe male factor infertility and/or previous implantation failures. The objective of this prospective randomized study was to compare the clinical outcome of 87 intracytoplasmic morphologically selected sperm injection (IMSI) cycles with 81 conventional intracytoplasmic sperm injection (ICSI) cycles in an unselected infertile population. IMSI did not provide a significant improvement in the clinical outcome compared with ICSI although there were trends for higher implantation (28.9% versus 19.5%), clinical pregnancy (54.0% versus 44.4%) and live birth rates (43.7% versus 38.3%) in the IMSI group. However, severe male factor patients benefited from the IMSI procedure as shown by significantly higher implantation rates compared with their counterparts in the ICSI group (29.6% versus 15.2%, P=0.01). These results suggest that IMSI may improve IVF success rates in a selected group of patients with male factor infertility. New technological developments enable the real time examination of motile spermatozoa with an inverted light microscope equipped with high-power differential interference contrast optics, enhanced by digital imaging. High magnification (over ×6000) provides the identification of spermatozoa with a normal nucleus and nuclear content. Intracytoplasmic injection of spermatozoa selected according to fine nuclear morphology under high magnification may improve the clinical outcome in cases with severe male factor infertility.

Hysteroscopic treatment of intrauterine adhesions is safe and effective in the restoration of normal menstruation and fertility

OBJECTIVE To assess the safety and efficacy of hysteroscopic adhesiolysis in patients with recurrent pregnancy loss and infertility. DESIGN Retrospective case report series. SETTING The obstetrics and gynecology clinic of a medical school. PATIENT(S) Forty women with recurrent pregnancy loss or infertility resulting from intrauterine adhesions. INTERVENTION(S) Hysteroscopic adhesiolysis in patients with recurrent pregnancy loss and infertility. MAIN OUTCOME MEASURE(S) Postoperative adhesion formation, intraoperative complication, conceivement after surgery, pregnancy rate, and pregnancies resulted in term or viable preterm infants. RESULT(S) Most patients with minimal or moderate adhesions were free of adhesions when compared with postoperative control subjects. However, adhesion re-formation was noted in 60% of the patients who initially had severe adhesions. Normal menstrual flow was restored in 81% of the patients. All the patients who had recurrent pregnancy loss conceived after treatment, and 71% of the pregnancies resulted in a term or viable preterm infant. Of the 16 infertile patients treated, 10 (63%) conceived and 6 (37%) were delivered of viable infants. CONCLUSION(S) Hysteroscopic adhesiolysis is a safe and effective procedure for restoring the normal menstrual pattern and fertility. The initial severity of the adhesions appears to correlate best with the reproductive outcome.

Recurrent implantation failure in assisted reproduction: how to counsel and manage. A. General considerations and treatment options that may benefit the couple

Recurrent implantation failure is a distressing phenomenon, both for the infertile couple and for the physician responsible for their treatment. Aetiology is often not clear and treatment options are vague. Particularly when transferred embryos are of good quality, recurrent implantation failure may be attributed to less than optimal embryo transfer technique, pathological lesions of the uterine cavity, the presence of hydrosalpinges, fibroids and endometriosis. Poor embryo quality, especially when repetitive, is a major impediment to successful implantation and cannot be corrected at the present time. Molecular abnormalities at the endometrial level and abnormal embryo-endometrium dialogue may be responsible for some cases of recurrent implantation failure. Furthermore, there may be over- or under-expressed genes that may be related to successful implantation. At the present time, the physician confronted with a couple presenting with recurrent implantation failure should discuss openly the potential causes of this phenomenon, with special emphasis on correctable causes, and offer remedies that are evidence based.

Luteal phase empirical low molecular weight heparin administration in patients with failed ICSI embryo transfer cycles: a randomized open-labeled pilot trial

BACKGROUND The pathology underlying recurrent implantation failures (RIF) is not clear and treatment options proposed are generally not evidence based. Although the effect of heparin on trophoblast biology has not been studied extensively, given the available data suggesting a possible beneficial effect of heparin on embryo implantation, we decided to undertake this pilot study. METHODS One hundred and fifty women with > or =2 failed assisted reproduction treatment cycles were included in this randomized open-label pilot trial. Participants underwent controlled ovarian stimulation with the long protocol and were randomly allocated to receive 1 mg/kg/day low molecular weight heparin (LMWH) or no treatment in addition to routine luteal phase support (LPS) on the day after oocyte retrieval. LPS and LMWH was continued up to the 12th gestational week in pregnant participants. RESULTS There were 26 (34.7%) live births in the LMWH group, and 20 (26.7%) in the control group (absolute difference 8.0%, 95% CI -4.2 to 24.9%, P = 0.29). There were 34 (45.3%) and 29 (38.7%) clinical pregnancies in the LMWH and control groups, respectively (absolute difference 6.6%, 95% CI -9.0 to 21.8%, P = 0.41). Implantation rates were 24.5 and 19.8% in the LMWH and control groups, respectively (absolute difference 4.7%, 95% CI -4.7 to 14.1%, P = 0.33). CONCLUSION Despite lack of statistical significance, observed relative increase by 30% in live birth rates with LMWH may be regarded as a clinically significant trend necessitating further research on the use of empirical LMWH in women with RIF and possibly in all women undergoing assisted reproduction treatment. Failure to demonstrate statistical significance of the observed treatment difference may be due to limited sample size of this pilot study.

Recurrent implantation failure in assisted reproduction: how to counsel and manage. B. Treatment options that have not been proven to benefit the couple

The success of assisted reproduction, although gradually increasing over the years, is still less than satisfactory. Many couples have benefited from this treatment; however, many have also been left frustrated following multiple failed attempts. Couples who fail to conceive after multiple IVF/intracytoplasmic sperm injection (ICSI) treatments often seek treatment options that are new and that have not been offered before. Some of these include immunological testing and treatment, allogenic lymphocyte therapy, intratubal transfer of zygotes and embryos, blastocyst transfer, sequential embryo transfer, assisted hatching, co-cultures, and preimplantation genetic screening for aneuploidy. Although the evidence behind some of these is more robust, most suffer from lack of well designed randomized trials comparing them with other treatment options. Randomized studies are extremely difficult to conduct, as couples will resist being randomized into a treatment group where previously failed procedures will be repeated. In the mean time, assisted reproduction programmes should resist offering treatment options that are not evidence based, or at least they should share with the couple the information that is available and should stress that none of these is a panacea for their problem.