In vitro fertilization does not increase the incidence of de novo copy number alterations in fetal and placental lineages.

Although chromosomal instability (CIN) is a common phenomenon in cleavage-stage embryogenesis following in vitro fertilization (IVF)1-3, its rate in naturally conceived human embryos is unknown. CIN leads to mosaic embryos that contain a combination of genetically normal and abnormal cells, and is significantly higher in in vitro-produced preimplantation embryos as compared to in vivo-conceived preimplantation embryos4. Even though embryos with CIN-derived complex aneuploidies may arrest between the cleavage and blastocyst stages of embryogenesis5,6, a high number of embryos containing abnormal cells can pass this strong selection barrier7,8. However, neither the prevalence nor extent of CIN during prenatal development and at birth, following IVF treatment, is well understood. Here we profiled the genomic landscape of fetal and placental tissues postpartum from both IVF and naturally conceived children, to investigate the prevalence and persistence of large genetic aberrations that probably arose from IVF-related CIN. We demonstrate that CIN is not preserved at later stages of prenatal development, and that de novo numerical aberrations or large structural DNA imbalances occur at similar rates in IVF and naturally conceived live-born neonates. Our findings affirm that human IVF treatment has no detrimental effect on the chromosomal constitution of fetal and placental lineages.

rs10732516 polymorphism at the IGF2/H19 locus associates with genotype-specific effects on placental DNA methylation and birth weight of newborns conceived by assisted reproductive technology.

Background: Assisted reproductive technology (ART) has been associated with low birth weight of fresh embryo transfer (FRESH) derived and increased birth weight of frozen embryo transfer (FET)-derived newborns. Owing to that, we focused on imprinted insulin-like growth factor 2 (IGF2)/H19 locus known to be important for normal growth. This locus is regulated by H19 imprinting control region (ICR) with seven binding sites for the methylation-sensitive zinc finger regulatory protein (CTCF). A polymorphism rs10732516 G/A in the sixth binding site for CTCF, associates with a genotype-specific trend to the DNA methylation. Due to this association, 62 couples with singleton pregnancies derived from FRESH (44 IVF/18 ICSI), 24 couples from FET (15 IVF/9 ICSI), and 157 couples with spontaneously conceived pregnancies as controls were recruited in Finland and Estonia for genotype-specific examination. DNA methylation levels at the H19 ICR, H19 DMR, and long interspersed nuclear elements in placental tissue were explored by MassARRAY EpiTYPER (n = 122). Allele-specific changes in the methylation level of H19 ICR in placental tissue (n = 26) and white blood cells (WBC, n = 8) were examined by bisulfite sequencing. Newborns' (n = 243) anthropometrics was analyzed by using international growth standards. Results: A consistent trend of genotype-specific decreased methylation level was observed in paternal allele of rs10732516 paternal A/maternal G genotype, but not in paternal G/maternal A genotype, at H19 ICR in ART placentas. This hypomethylation was not detected in WBCs. Also genotype-specific differences in FRESH-derived newborns' birth weight and head circumference were observed (P = 0.04, P = 0.004, respectively): FRESH-derived newborns with G/G genotype were heavier (P = 0.04) and had larger head circumference (P = 0.002) compared to newborns with A/A genotype. Also, the placental weight and birth weight of controls, FRESH- and FET-derived newborns differed significantly in rs10732516 A/A genotype (P = 0.024, P = 0.006, respectively): the placentas and newborns of FET-derived pregnancies were heavier compared to FRESH-derived pregnancies (P = 0.02, P = 0.004, respectively). Conclusions: The observed DNA methylation changes together with the phenotypic findings suggest that rs10732516 polymorphism associates with the effects of ART in a parent-of-origin manner. Therefore, this polymorphism should be considered when the effects of environmental factors on embryonic development are studied.

Short- and long-term health consequences and current satisfaction levels for altruistic anonymous, identity-release and known oocyte donors.

STUDY QUESTION: What are the short- and long-term health consequences and current satisfaction of altruistic oocyte donors? SUMMARY ANSWER: Donating oocytes in the majority (>90%) of donation cycles is not associated with harmful long-term general or reproductive health effects. WHAT IS KNOWN ALREADY: Knowledge of long-term health effects of oocyte donation on donors is sparse and follow-up studies have usually been carried out on commercial donors. Thus far, no major long-term harmful effects have been demonstrated. Most studies have reported a high level of donor satisfaction, but also less favorable experiences have been published. STUDY DESIGN, SIZE AND DURATION: A retrospective cross-sectional survey of all women who had donated oocytes between 1990 and 2012 at three fertility clinics was carried out in spring 2013. A self-administered questionnaire was sent out to a total of 569 former oocyte donors. PARTICIPANTS, SETTING, METHODS: In all, 428 past donors answered the questionnaire assessing donor's demographic characteristics, short- and long-term medical and psychological experiences and satisfaction related to donations. Of the donors, 87% (371/428) were unknown and 13% (57/428) were known to the recipient. The mean follow-up time after the donation was 11.2 years (range from 0.5 to 23 years) and the mean age of the respondents was 42 years at the time of the study. To learn whether the demographic profile of donors was affected by the Finnish Assisted Reproduction Technology (ART) Act of 2007, we divided the 428 respondents into two groups: (i) women whose first donation took place between 1990 and 2007 (79% of the respondents) and (ii) women whose first donation took place between 2008 and 2012 (21% of the respondents). Before 2008, donors were non-identifiable (anonymous) but after 2008 persons born as a result of gamete donation could, from the age of 18, receive on request information identifying the donor. MAIN RESULTS AND THE ROLE OF CHANCE: The response rate was 75% (428/569). The mean age of the donors did not differ between the two time periods, but there was a higher proportion of donors in the youngest age group (20-24 years) and more childless donors (P < 0.05) after 2008 than between 1990 and 2007. Immediate complications occurred in 7.2% (42/582) of the donation cycles and the most common complication was ovarian hyperstimulation syndrome (OHSS) in 5.0% (29/582) of the treatments. There were no reports of ovarian or uterine cancer and only one case of breast cancer. After the donation, 11.5% of the donors experienced unsuccessful attempts to become pregnant. Almost all donors (99%) were satisfied or very satisfied with their decision to donate and 95% would warmly recommend it to other women. There were no differences between the known and unknown donors in this respect, or between the two time periods (before or after the ART Act in 2008). Four donors (1%) had regretted donation, and 7% would have wanted to have more support before and 14% after the donation. LIMITATIONS, REASON FOR CAUTION: Although the response rate was high, 25% of all former donors in the three participating clinics could not be included due to lack of response. The results are based on self-reported assessment of the experiences of former donors, and it is not possible to estimate the influence of recall bias. WIDER IMPLICATIONS OF THE FINDINGS: To our knowledge, this is the largest study of health consequences and satisfaction levels on oocyte donors. Data from this study can be used to inform donor candidates about the medical aspects involved in the treatment and it provides information on how to support these women during and after the donation. STUDY FUNDING/COMPETING INTERESTS: This study was supported by grants from the Medical Society Life and Health, and from the Otto A. Malm Foundation. There are no competing interests to report.

In-vitro maturation: its role in fertility treatment.

PURPOSE OF REVIEW: In-vitro maturation of oocytes was primarily developed to make in-vitro fertilization safer and simpler for women with polycystic ovaries and high risk of ovarian hyperstimulation syndrome. In-vitro maturation has potentially many advantages over conventional in-vitro fertilization. A simple protocol with decreased or no hormonal stimulation before oocyte retrieval and thus lower cost of the treatment cycle are clear benefits. More importantly, the risk of ovarian hyperstimulation syndrome is entirely avoided. RECENT FINDINGS: The clinical outcome has substantially improved in recent years with pregnancy rates between 20 and 54%. The birth of approximately 400 children has been reported in the literature, but about three times as many are known to have been born. The postnatal follow-up studies of the children have been reassuring. SUMMARY: Although good results have been reported by some clinics, in-vitro maturation has not yet become a mainstream fertility treatment. The most important reason for this is the lower chance of a live birth per treatment compared with conventional in-vitro fertilization. Several aspects of the clinical in-vitro maturation need to be improved to give in-vitro maturation a place among assisted reproduction techniques it deserves.

In-vitro maturation of eggs: is it really useful?

In in-vitro maturation (IVM), immature oocytes are collected from small antral follicles and allowed to mature in the laboratory before routine in-vitro fertilization or micro-injection. The authors' experience in IVM is based on the treatment of two main groups of patients: women with polycystic ovaries and women with normal ovaries. Patients with polycystic ovarian syndrome have irregular, mostly anovulatory cycles and are at increased risk for ovarian hyperstimulation syndrome because of their higher sensitivity to gonadotropins. Women with normal ovarian function may wish to avoid the side-effects of hormone injections, and therefore IVM has also been offered to couples with tubal, male factor and unexplained infertility. In all these groups of patients, immature oocytes have successfully been matured, fertilized and embryos transferred. Pregnancy rates have been reported to be between 4% and 54%. More than 300 children have been born and follow-up studies have reported no major concerns about the pregnancies, deliveries or health of the babies. There are still many questions concerning IVM. As the factors regulating follicle selection are poorly understood, no specific markers for the optimal time of immature oocyte collection have been defined. Furthermore, basic knowledge on the complex intracellular processes involved in the cytoplasmic maturation of human oocyte is lacking, making the design of optimal culture conditions for maturation difficult. The possible long-term effects of IVM on the health and development of children needs future study.

Frozen embryo transfers: implications of clinical and embryological factors on the pregnancy outcome.

BACKGROUND: Frozen embryo transfers are characterized by impaired pregnancy outcome and increased incidence of pregnancy loss as compared with fresh IVF/ICSI embryo transfers. In this study, we performed a retrospective analysis of clinical and embryological factors that potentially influence the outcome of frozen embryo transfer. METHODS: We reviewed the outcome of 1242 frozen embryo transfers with respect to the age of the woman, the method of fertilization, embryo quality before and after freezing and the number of embryos transferred. RESULTS AND CONCLUSIONS: The pregnancy (positive hCG) and clinical pregnancy rates were 25.8 and 21.1%, respectively. A total of 107 (33.3%) of the 321 pregnancies identified by a positive hCG test miscarried either before (18.4%) or after (15%) the clinical recognition of gestational sac(s). The delivery rate for the frozen embryo transfers analysed was 17.2%. Our data revealed that the delivery rate after frozen embryo transfer was dependent on both the woman's age and the quality of embryos transferred, at the same time being unaffected by IVF/ICSI treatment. In addition, the increased woman's age at IVF/ICSI treatment was identified as the only parameter elevating the biochemical pregnancy rate, whereas the clinical abortion rate was found to be unrelated to the clinical or embryological parameters studied.

Obstetric and perinatal outcome and preliminary results of development of children born after in vitro maturation of oocytes.

BACKGROUND: Careful follow-up of children born after in vitro maturation (IVM) of human oocytes is essential because the technique is still very new. METHODS: Obstetric and perinatal data were collected from all deliveries after IVM treatment during 1999-2004. The growth and development of IVM children was assessed at 6, 12 and 24 months using Muenchener Funktionelle Entwicklungs Diagnostik and Bayley Scales of Infants. RESULTS: In total, 43 women [age 31.2 +/- 3.9 (mean +/- SD) years] gave birth to 40 singleton infants and three sets of twins (multiple rate 7.0%). Obstetric complications occurred in 15 pregnancies (35%). The mean birthweight of singleton infants was 3550 +/- 441 g and that of twins 2622 +/- 194 g. The rate of preterm birth infants was 5% in singletons. No perinatal deaths occurred. At the age of 12 months, eight children (19%) expressed minor developmental problems and one girl was found to have optical glioma. At 2 years of age, neuropsychological development was within the normal range. CONCLUSIONS: The obstetric and perinatal outcome was good, and the mean birthweight of the infants was normal. Minor developmental delay was overexpressed at 12 months, but the development of the children was normal at 2 years.

Favourable pregnancy results with insemination of in vitro matured oocytes from unstimulated patients.

BACKGROUND: The purpose has been to develop an in vitro oocyte maturation (IVM) method for a wide range of patients. METHODS: A total of 239 cycles with immature oocyte retrieval (IOC) were carried out without hormonal priming. Patients with regular cycles and normal or polycystic ovaries (PCO) and anovulatory cycles with PCOS were included. Insemination or intracytoplasmic sperm injection (ICSI) according to sperm quality was alternatively used in fertilization of the matured oocytes. RESULTS: A total of 971 immature oocytes (mean 8.0 +/- 5.2) were collected in 122 IVM-IVF cycles and 851 oocytes (mean 7.3 +/- 4.4) in 117 IVM-ICSI cycles. The oocyte maturation and fertilization rate was 62.6% and 37.7% after insemination, and 53.9% and 69.3% after ICSI, respectively. The mean number of embryos transferred was 1.6. Clinical pregnancy rate per IOC was 23.8% in IVM-IVF and 17.1% in IVM-ICSI (ns). Implantation rate was higher in IVM-IVF (24.2%) than in IVM-ICSI (14.8%) (P < 0.05). CONCLUSIONS: Insemination of IVM oocytes functions well, resulting in comparable pregnancy rates per IOC between IVM-IVF and IVM-ICSI. Good pregnancy results can be achieved both in patients with regular cycles and with PCO(S) by transferring only one or two embryos at a time.

Elevated incidence of chromosomally chaotic embryos among frozen-thawed preimplantation embryos.

OBJECTIVE: The aim of the study was to evaluate the effect of cryopreservation on the formation of chromosomal abnormalities in human preimplantation embryos. STUDY DESIGN: The chromosomal constitutions of cleavage stage embryos (n = 61) were assessed using fluorescent in situ hybridisation (FISH) technique, applying probes for chromosomes 13, 16, 18, 21, X and Y. Study group embryos frozen at zygote or two-cell stage (n = 29) were cultured in vitro post-thawing until they reached four- to six-cell stage, after which their chromosomal constitutions were assessed. Control group embryos frozen at four- to six-cell stage (n = 32) were analysed immediately after thawing in order to exclude any post-thaw effect. The proportions of genetically normal and abnormal embryos were compared between study and control group. RESULTS: The proportions of normal, aneuploid and mosaic embryos were similar in both groups. However, significantly (P < 0.05) higher proportion of chaotic embryos in study (24.1%) compared to control group (6.3%) was observed. CONCLUSION: The elevated level of chromosomally chaotic embryos among embryos that had undergone cellular division after thawing as compared to embryos analysed immediately after thawing indicates a potential negative impact of cryopreservation on the formation of chromosomal abnormalities in preimplantation embryos.

Recombinant LH is equally effective as recombinant hCG in promoting oocyte maturation in a clinical in-vitro maturation programme: a randomized study.

BACKGROUND: Fertilization treatment using oocytes matured in vitro from pre-ovulatory follicles has many potential applications. It minimizes the risk of severe ovarian hyperstimulation and is an alternative for women with polycystic ovary syndrome who may have problems regarding stimulation for IVF. In-vitro maturation (IVM) may prove important for subjects needing fertility preservation, and also provides information about the final stages of oocyte maturation. METHODS: From a randomized study of 73 women in an IVF programme, 36 subjects with 228 oocytes were allocated for oocyte maturation in culture medium with recombinant hCG, and 37 subjects with 256 oocytes for maturation with recombinant LH. The primary outcome was the rate of nuclear maturation of oocytes to metaphase II. During the same period, 32 women outside the study underwent 38 individually tailored IVM treatments. RESULTS: The oocyte maturation rate was 54.8% with hCG and 55.9% with LH; fertilization and cleavage rates were not significantly different. Three pregnancies were achieved in the hCG group and one in the LH group. Seven pregnancies (22.6% per embryo transfer) were achieved in the parallel group. CONCLUSIONS: Recombinant hCG or LH are equally effective in promoting oocyte maturation in a clinical IVM programme.

Elective single embryo transfer yields good delivery rates in oocyte donation.

BACKGROUND: High pregnancy rates have been noted after oocyte donation (OD). Multiple pregnancies should be avoided, because oocyte recipients have an increased risk of obstetric complications. METHODS: We analysed our OD results from 2000-2001 when elective single embryo transfer (eSET) was introduced as a recommended policy for all recipients if at least one good quality embryo was available. The results were compared with those achieved in 1998-1999, when usually two embryos were transferred (double embryo transfer, DET). Between 1998 and 2001, 100 healthy women donated oocytes and 135 fresh embryo transfers were carried out. The mean age of the donors was 31 years and that of the recipient women was 35 years. RESULTS: The proportion of eSET of all OD transfers was 17.1% in 1998-1999 and 61.0% in 2000-2001. There was no statistically significant difference in clinical pregnancy (36.8 versus 45.8%) and delivery rates (31.6 versus 33.9%) per embryo transfer between the two time periods. The proportion of twins declined from 29% (1998-1999) to 10% (2000-2001). The delivery rate was similar after eSET and DET (32.6 versus 32.1% respectively). CONCLUSIONS: By increasing the proportion of eSETs it is possible to reduce the number of twins without affecting delivery rates in oocyte recipients.

Effect of developmental stage of embryo at freezing on pregnancy outcome of frozen-thawed embryo transfer.

BACKGROUND: The study aim was to investigate the impact of the developmental stage of embryos on pregnancy outcome of frozen embryo transfer (FET). METHODS: The survival rates of embryos after thawing and pregnancy outcome following FET were compared retrospectively between three cryopreservation strategies utilizing either zygote, day 2 or day 3 embryo freezing. RESULTS: A total of 4006 embryos was analysed in 1657 thaw cycles. The highest (P < 0.0001) survival rate (all cells survived) was observed for zygotes (86.5%), followed by day 2 (61.7%) and day 3 (43.1%) embryos. FET was performed in 1586 (95.7%) of all thaw cycles, resulting in overall clinical pregnancy and implantation rates of 20.7 and 14.2% respectively. The delivery rate per transfer was 16.5%, and live birth rate per transferred embryo 11%. There were no significant differences in clinical pregnancy, implantation, delivery and birth rates between frozen zygote, day 2 and 3 embryo transfers. However, an elevated miscarriage rate was observed in the day 3 group (45%) compared with zygotes (21.3%; P = 0.049) and day 2 embryos (18.3%; P = 0.004). The overall efficacy of FET (birth rate per thawed embryo) was 7.3%. The efficacy was lower in day 3 group (4.2%) than in the zygote (7.1%; P = 0.082) and day 2 (7.6%; P = 0.027) groups. CONCLUSIONS: The developmental stage of embryos at freezing has a profound effect on their post-thaw survival, but seems to have little effect on rates of clinical pregnancy, implantation, delivery and birth after FET. The elevated miscarriage rate for day 3 frozen embryo transfers may be caused by damage during freeze-thaw procedures. The low survival rate and elevated miscarriage rate were both responsible for a reduced overall efficacy for day 3 FET when compared with zygotes and day 2 embryos.

Early cleavage predicts the viability of human embryos in elective single embryo transfer procedures.

BACKGROUND: The reduction of multiple pregnancies by using elective single embryo transfers (eSET) requires critical and careful selection of the embryo for transfer. The current study was undertaken to assess whether early cleavage could be used as a marker of embryo competence in eSET procedures. METHODS: The study included analysis of 178 eSET procedures. All embryos were checked for early cleavage at 25-27 h post insemination or ICSI. The embryos that possessed two cells at 25-27 h post insemination or ICSI were designated as 'early cleavage' (EC) embryos and those that had not yet cleaved were classified as 'no early cleavage' (NEC) embryos. Selection of the embryo for transfer was based on embryo morphology and growth rate on day 2 and not early cleavage. Clinical parameters were compared between 72 EC and 106 NEC single embryo transfers. RESULTS: A significantly higher clinical pregnancy rate was observed after transfer of EC (50%) than NEC (26.4%) embryos. CONCLUSIONS: The current study provides compelling evidence that EC embryos possess significantly higher developmental competence than NEC embryos.

Influence of oocytes and spermatozoa on early embryonic development.

OBJECTIVE: To evaluate the effect of oocytes and spermatozoa on early embryonic development. DESIGN: Retrospective study. SETTING: Infertility Clinic, the Family Federation of Finland. PATIENT(S): Fifty-nine oocyte donation cycles with oocytes shared among 118 recipient couples. INTERVENTION(S): Culture of all fertilized oocytes. MAIN OUTCOME MEASURE(S): Standard sperm (concentration, progressive motility, and morphology according to Tygerberg strict criteria) and embryo (morphology and cleavage stage) characteristics. RESULT(S): A marked effect of the oocyte on both embryo morphology and blastomere cleavage rate was demonstrated. In addition, a significant sperm effect on blastomere cleavage rate was found. Sperm morphology as determined according to strict criteria rather than sperm count or progressive motility was positively associated with the blastomere cleavage rate. None of the measured sperm characteristics influenced embryo morphology. CONCLUSION(S): Embryo morphology, i.e., fragmentation and blastomere uniformity, are predominantly determined by oocyte quality, whereas both the oocyte and spermatozoa influence the blastomere cleavage rate.