Possible risk for cancer among children born following assisted reproductive technology in Israel.

BACKGROUND: Among children conceived by assisted reproductive technology (ART), increased risk of adverse birth outcomes has been observed, including multiple births, preterm births, and congenital malformations. Regarding cancer among ART-conceived children, findings are discrepant. METHODS: This is a historical cohort of 9,042 ART-conceived children and 211,763 spontaneously conceived (SC) children born from 1997 through 2004. The median duration of follow-up was 10.6 years (interquartile range 9.0-12.3) in the ART group and 9.3 years (interquartile range 8.0-10.6) in the SC group. The cohort database was linked with the Israel National Cancer Registry updated until December 31, 2011 using each child's personal identification number. RESULTS: Twenty-one cases of cancer were identified in the ART group (2.2 per 10,000 person-years), as compared to 361 cancer cases in the SC group (1.8 per 10,000 person-years). The relative risk (RR) for overall cancer in the ART group compared to the SC group adjusted for maternal characteristics was 1.18 (95% confidence interval [CI] 0.80-1.75). ART children had a significantly increased risk for specific cancers, although based on small number of cases, including two cases of retinoblastoma (RR 6.18, 95% CI 1.22-31.2), as well as four cases of renal tumors (RR 3.25, 95% CI 1.67-6.32). CONCLUSION: A statistically significant increased risk for two pediatric cancers was found. However, for overall types of cancer the risk estimate was elevated but not statistically significant. Further studies with larger sample size and longer follow-up time are warranted in order to either confirm or refute these findings.

How Many Embryos should be Transferred? The Relevance of Parity and Obstetric History.

BACKGROUND: Fertility treatments are responsible for the rise in high order pregnancies in recent decades and their associated complications. Reducing the number of embryos returned to the uterus will reduce the rate of high order pregnancies. OBJECTIVES: To explore whether obstetric history and parity have a role in the clinician's decision making regarding the number of embryos transferred to the uterus during in vitro fertilization (IVF). METHODS: In a retrospective study for the period August 2005 to March 2012, data of twin deliveries > 24 weeks were collected, including parity, mode of conception (IVF vs. spontaneous), gestational age at delivery, preeclampsia, birth weight, admission to the neonatal intensive care unit (NICU), and Apgar scores. RESULTS: A total of 1651 twin deliveries > 24 weeks were record- ed, of which 959 (58%) were at term (> 37 weeks). The early preterm delivery (PTD) rate (< 32 weeks) was significantly lower with increased parity (12.6%, 8.5%, and 5.6%, in women with 0, 1, and ≥ 2 previous term deliveries, respectively). Risks for PTD (< 37 weeks), preeclampsia and NICU admission were significantly higher in primiparous women compared to those who had one or more previous term deliveries. Primiparity and preeclampsia, but not IVF, were significant risk factors for PTD. CONCLUSIONS: The risk for PTD in twin pregnancies is significantly lower in women who had a previous term delivery and decreases further after two or more previous term deliveries. This finding should be considered when deciding on the number of embryos to be transferred in IV.

Ovarian stimulation for oocyte cryopreservation for prevention of age-related fertility loss: one in five is a low responder.

Oocyte cryopreservation for age-related fertility loss is gaining interest considering the tendency to postpone motherhood in many societies. Little is currently known about the actual efficiency of this approach. We aimed to explore ovarian response of presumably fertile women undergoing in vitro fertilization for this indication. A total of 105 women underwent 151 stimulation cycles at mean age 37.7 ± 2.4. None had known infertility. Mean daily starting FSH dose was 371 ± 110 (225-600). Mean number of mature oocytes cryopreserved at the first completed cycle was 9.7 ± 7.5 (0-43). However, 21% of started cycles were either cancelled before egg retrieval or resulted in 0-3 mature oocytes retrieved. Therefore, women considering oocyte cryopreservation for prevention of age-related fertility decline should be encouraged to perform this procedure at younger age than, preferably before 35.

Protease-activated-receptor 1 polymorphisms correlate with risk for unexplained recurrent pregnancy loss: a pilot study querying an association beyond coagulation.

BACKGROUND: Recurrent pregnancy loss (RPL) has been associated with impaired maternal-fetal communication. Protease-activated-receptor 1 (PAR1) is critical for trophoblast invasion and establishment unrelated to its role in vascular biology. OBJECTIVES: To analyze whether polymorphisms of PAR1 [-1426C/T], [-506I/D], and/or IVS[-14A/T] are associated with unexplained RPL. PATIENTS/METHODS: A case-control pilot study conducted in 39 healthy women with history of unexplained RPL and 98 women with a full-term, uncomplicated deliveries and no history of RPL. RESULTS: Women with RPL were significantly more likely to be heterozygous for [-1426C/T] (12.8% versus 3.2%; p=0.049); the heterozygous state for IVS[-14A/T] was also more common (15.4% versus 4.4%; p=0.064). There was no difference between groups for [-506I/D] genotypes. The functional consequence for [-1426C/T] and IVS[-14A/T] polymorphisms is underscored by the markedly low PAR1 mRNA levels in those women. Bioinformatics indicate generation of a new consensus motif for repressor Kruppel-like factor 3 (KLF3) in [-1426T]. Moreover, chromatin immunoprecipitation (ChIP) analysis confirmed a physical association between KLF3 protein and the hPar1 DNA obtained from women with the [-1426C/T] polymorphism. CONCLUSIONS: We hypothesize that the significantly low PAR1 levels impact placenta establishment and consequently pregnancy outcome, thereby profiling a novel risk factor for unexplained RPL.

The dynamics of serum anti-Mullerian-hormone levels during controlled ovarian hyperstimulation with GnRH-antagonist short protocol in polycystic ovary syndrome and low responders.

OBJECTIVE: To determine whether the decrease in AMH levels during ovarian hyperstimulation for IVF occurs in patients with polycystic ovary syndrome (PCOS) and patients with low ovarian reserve (LOR), as in normal cycling women. STUDY DESIGN: A cohort of 22 infertile patients treated in a single tertiary center with a GnRH-antagonist short protocol for IVF were prospectively included and divided into three groups: PCOS with hyperandrogenism (n=7), LOR (n=8) and control (n=7). Serum AMH levels were measured before and during FSH treatment, on the day of HCG administration, at the mid-luteal phase, and 14 days after embryo transfer. The three groups were compared using an ANOVA model in the case of continuous data and with Fisher's exact test when the data were discrete. RESULTS: In the PCOS group, AMH levels increased at the beginning of the stimulation, but later decreased, until the mid-luteal stage. In the other two groups, AMH levels decreased throughout ovarian stimulation until the mid-luteal stage. In all groups, AMH levels returned to baseline levels two weeks after HCG administration, regardless of treatment outcome (pregnancy or not). CONCLUSIONS: AMH levels decline during controlled ovarian hyperstimulation with a GnRH-antagonist short protocol in women with low and normal ovarian reserves. In contrast, in women with PCOS, an increase in AMH levels precedes this decline. These findings may support the hypothesis that androgens may play a role in AMH regulation in women.

Preimplantation genetic diagnosis in genomic regions with duplications and pseudogenes: long-range PCR in the single-cell assay.

Long-range PCR is generally employed for the analysis of disease-causing mutations in genes with homologous pseudogene copies. However, long-range PCR is challenging when performed on single cells, as in preimplantation genetic diagnosis (PGD) of monogenic disorders. PGD on single cells requires concurrent analysis of a mutation together with multiple linked polymorphic markers from closely related family members to prevent misdiagnosis. In PGD cases involving childless de novo mutation carriers, linkage cannot be performed based on family members but rather must first be identified in single gametes. This can be an especially difficult task if the mutation to be assayed lies in a duplicated genomic region because gene-specific long-range PCR must be coupled with short-range PCR analysis of genetic markers on single cells. Here, we describe a novel method by which accurate PGD of pseudogene-homologous mutations can be achieved. Essentially, we performed whole genome amplification on single sperm or blastomeres followed by haplotype construction and long-range PCR-based mutation analysis. This original and universal strategy was used to establish allelic association for two different mutations in genes with one or more pseudogene copies (IKBKG and PKD1). The method was also sensitive enough to detect unexpected germline mosaicism in one mutation carrier.

DHEA supplementation may improve IVF outcome in poor responders: a proposed mechanism.

OBJECTIVE: Dehydroepiandrosterone (DHEA) supplementation for poor responders may improve ovarian response and IVF treatment outcome. This study aimed to determine the mechanism of action of DHEA, and specifically, the stage of folliculogenesis influenced by DHEA. STUDY DESIGN: This is a prospective, self-controlled study of poor responders to IVF treatment, comparing day 3 biochemical (anti-Mullerian hormone (AMH), inhibin B and FSH) and ultrasound (antral follicle count (AFC)) ovarian reserve markers and IVF treatment outcome before and after DHEA supplementation of at least 3 months duration. RESULTS: Thirty-two women were included. Following DHEA, there was a significant increase in AFC (P=0.0003) without significant changes in the baseline biochemical parameters AMH, inhibin B, or FSH. The enhanced response comprised increased peak estradiol levels (P=0.0005), number of follicles >15 mm, oocytes, MII oocytes and embryos (P=0.004, P=0.00001, P=0.0004 and P=0.0006, respectively) and oocytes number/total FSH dose (P=0.0009). The proportion of cancelled cycles due to very poor response decreased significantly (P=0.02). CONCLUSIONS: DHEA does not appear to exert influence via recruitment of pre-antral or very small antral follicles (no change in AMH and inhibin B) but rather by rescue from atresia of small antral follicles (increased AFC).

PGD for germline mosaicism.

The aim of this study was to develop and perform a preimplantation genetic diagnosis (PGD) assay discriminating between wild-type and mutant alleles in two families with germline mosaicism. Family 1 had two children affected with severe myoclonic epilepsy (SCNA1A del exons 1-22). Family 2 had two children with tuberous sclerosis (TSC2 C1327T) and two healthy children. Neither mutation was detected in genomic DNA derived from the parents in either family. Informative microsatellite markers flanking SCNA1A and TSC2 along with the identified mutations were used to construct haplotypes. For tuberous sclerosis, single spermatozoa were analysed using a multiplex assay that included six informative markers and the TSC2 mutation. In family 1, deletion in the maternal allele was detected in the affected child. In family 2, both affected children and one healthy child shared the same paternal allele. To confirm mutant paternal transmission, single spermatozoa were analysed for the mutation along with six markers. Of 44 single spermatozoa, four showed the mutant T allele, allowing linkage between the mutation and the genetic markers. Both families delivered healthy children following IVF/PGD. In conclusion, germline mosaicism complicates allele assignment when constructing haplotypes for PGD. Sperm analysis is a useful tool for verifying allelic linkage.

Preimplantation genetic diagnosis for fetal neonatal alloimmune thrombocytopenia due to antihuman platelet antigen maternal antibodies.

OBJECTIVE: To develop a reliable preimplantation genetic diagnosis protocol for antihuman platelet antigen-1 incompatibility for a family in whom antenatal treatment was not possible because of the mother's hypersensitivity to intravenous immunoglobulin (IVIG). METHODS: Haplotypes were constructed from genomic DNA of the family members. A polymerase chain reaction protocol that included eight microsatellite polymorphic markers and the ITGB3-specific (T196C, rs5918) polymorphism were multiplexed to be used in a single cell protocol, and single blastomeres were analyzed. RESULTS: In one preimplantation genetic diagnosis cycle, out of 28 retrieved oocytes, 24 embryos fertilized and 12 underwent biopsy. Three embryos were found to be antihuman platelet antigen-1b/1b homozygotes and two were transferred. This cycle resulted in an uneventful pregnancy and birth of a healthy child. CONCLUSION: In cases in which there is antihuman platelet antigen incompatibility and IVIG cannot be administered, preimplantation genetic diagnosis is a reliable alternative to enable birth of unaffected children.

Prevention of lysosomal storage diseases and derivation of mutant stem cell lines by preimplantation genetic diagnosis.

Preimplantation genetic diagnosis (PGD) allows birth of unaffected children for couples at risk for a genetic disorder. We present the strategy and outcome of PGD for four lysosomal storage disorders (LSD): Tay-Sachs disease (TSD), Gaucher disease (GD), Fabry disease (FD), and Hunter syndrome (HS), and subsequent development of stem cell lines. For each disease, we developed a family-specific fluorescent multiplex single-cell PCR protocol that included the familial mutation and informative markers surrounding the mutation. Embryo biopsy and PGD analysis were performed on either oocytes (polar bodies one and two) or on single blastomeres from a six-cell embryo. We treated twenty families carrying mutations in these lysosomal storage disorders, including 3 couples requiring simultaneous analysis for two disorders (TSD/GD, TSD/balanced Robertsonian translocation 45XYder(21;14), and HS/oculocutaneus albinism). These analyses led to an overall pregnancy rate/embryo transfer of 38% and the birth of 20 unaffected children from 17 families. We have found that PGD for lysosomal disorders is a safe and effective method to prevent birth of affected children. In addition, by using mutant embryos for the derivation of stem cell lines, we have successfully established GD and HS hESC lines for use as valuable models in LSD research.

Preimplantation genetic diagnosis (PGD)--prevention of the birth of children affected with endocrine diseases.

OBJECTIVE: To develop a reliable and accurate preimplantation genetic diagnosis (PGD) method in six families with endocrine diseases: persistent hyperinsulinemic hypoglycemia of infancy (PHHI), congenital adrenal hyperplasia (CAH) salt-wasting form, Sanjat-Sakati syndrome and multiple endocrine neoplasia 2A (MEN 2A). METHODS: For each disease a battery of at least four informative markers surrounding the tested gene were identified and for each family a protocol of multiplex fluorescent markers was developed and performed on single cells. RESULTS: PGD for PHHI was performed in three families. In family 1 two healthy children were born from different cycles, in family 2 three healthy children were born from two cycles, and in family 3 a healthy boy was born. For CAH in one family a healthy girl was born. One PGD cycle for Sanjat-Sakati resulted in a clinical pregnancy that was terminated due to high nuccal translucency (46X0). For one family with MEN 2A disease, the eighth PGD cycle resulted in birth of healthy twins. In all children genetic confirmation of the healthy status was performed. CONCLUSIONS: PGD is an effective method for preventing birth of affected children with endocrine disorders. Increasing the awareness of clinicians to the availability of these methods is most important.

Preventing mucopolysaccharidosis type II (Hunter syndrome): PGD and establishing a Hunter (46, XX) stem cell line.

OBJECTIVES: Preimplantation genetic diagnosis (PGD) enables the identification of affected embryos prior to implantation. We present for the first time three families in which either the oocytes or embryos obtained from female carriers of mutations in the iduronate-2-sulfatase (IDS) gene underwent PGD for mucopolysaccharidosis type II (Hunter syndrome). Furthermore, we report the first ever derivation of a Hunter's syndrome (46, XX) human stem cell line from embryos (HESC) carrying the IDS and oculocutaneus albinism type 2 mutations. METHODS: Combined polar body (PB) 1 and 2 or a single cell of a six- to eight-cell embryo (blastomere) was used for genetic analysis by multiplex polymerase chain reaction assay using six microsatellite polymorphic markers flanking the gene and mutation. RESULTS: One couple underwent four PB-PGD cycles, with birth of a healthy girl; the second couple with one PB-PGD cycle had healthy twins; the third couple underwent seven cycles of double PGD for Hunter and Albinism syndrome with birth of healthy twins. One novel Hunter 46, XX HESC line was established displaying typical characteristics of HESC cells. CONCLUSIONS: PGD is a reliable method to prevent pregnancy of children affected with Hunter syndrome. In addition, derived HESC can be further utilized for drug testing and better understanding of the pathogenesis of this syndrome.

Age-related normograms of serum antimüllerian hormone levels in a population of infertile women: a multicenter study.

OBJECTIVE: To produce age-related normograms for serum antimüllerian hormone (AMH) level in infertile women without polycystic ovaries (non-PCO). DESIGN: Retrospective cohort analysis. SETTING: Fifteen academic reproductive centers. PATIENT(S): A total of 3,871 infertile women. INTERVENTION(S): Blood sampling for AMH level. MAIN OUTCOME MEASURE(S): Serum AMH levels and correlation between age and different percentiles of AMH. RESULT(S): Age-related normograms for the 3rd, 10th, 25th, 50th, 75th, 90th, and 97th percentiles of AMH were produced. We found that the curves of AMH by age for the 3rd to 50th percentiles fit the model and appearance of linear relation, whereas the curves of >75th percentiles fit cubic relation. There were significant differences in AMH and FSH levels and in antral follicle count (AFC) among women aged 24-33 years, 34-38 years, and ≥39 years. Multivariate stepwise linear regression analysis of FSH, age, AFC, and the type of AMH kit as predictors of AMH level shows that all variables are independently associated with AMH level, in the following order: AFC, FSH, type of AMH kit, and age. CONCLUSION(S): Age-related normograms in non-PCO infertile women for the 3rd to 97th percentiles were produced. These normograms could provide a reference guide for the clinician to consult women with infertility. However, future validation with longitudinal data is still needed.

Preimplantation genetic diagnosis (PGD) for a treatable disorder: Gaucher disease type 1 as a model.

BACKGROUND: Preimplantation genetic diagnosis (PGD) is a technique that enables identification of unaffected embryos prior to in vitro fertilization (IVF) transfer in couples at risk for a Mendelian disorder. Most cases involve severe genetic diseases with neurological features and/or major malformations. We present two couples in which PGD was performed for prevention of type 1 Gaucher disease, a non-neuronopathic, non-lethal disorder. MATERIALS AND METHODS: We developed a multiplex fluorescent PCR protocol, simultaneously amplifying the familial mutations and eight closely spaced, highly polymorphic informative microsatellite markers surrounding the gene, to be used for PGD analysis. RESULTS: Couple #1 mother was homozygous for the N370S mutation and the father carried the 84GG mutation; their first daughter receives specific Gaucher therapy. One PGD cycle resulted in seven embryos of which four had the paternal wild type allele; two were transferred resulting in a healthy baby boy born at term. Couple #2, each a carrier (N370S and R359Q), whose first-born child had died (age 5years) of Gaucher disease, underwent 7 PGD cycles. Only one cycle resulted in a clinical pregnancy but a miscarriage was followed at 10weeks. CONCLUSIONS: PGD is an effective and accurate method for preventing Gaucher disease type I in carrier couples. Since this disease is treatable, special ethical considerations and careful selection of couples should be performed.

PGD for fragile X syndrome: ovarian function is the main determinant of success.

BACKGROUND: PGD for fragile X syndrome (FRAX) is inefficient, probably owing to fewer oocytes, poor embryo quality and difficulties in genetic analysis. We investigated IVF-PGD in FRAX mutation carriers compared with controls, looking at the effects of oocyte and embryo number/quality on live birth outcome. METHODS: We performed IVF-PGD in 27 patients with the FRAX mutation and 33 controls with other genetic diseases. Genetic testing was by multiplex PCR. RESULTS: Seventy-nine and 108 IVF-PGD cycles were started in FRAX mutation carriers and controls, respectively. Twenty-two patients had a premutation (CGG repeat number 60-200) and five had a full mutation (300-2000 CGG repeats). FRAX patients required higher doses of gonadotrophins (6788 ± 2379 versus 4360 ± 2330, P< 0.001) but had lower peak serum estradiol levels (8166 ± 5880 versus 10 211 ± 4673, P = 0.03) and fewer oocytes retrieved (9.8 ± 6 versus 14 ± 8, P = 0.01). The cancellation rate (unsatisfactory ovarian response) was higher in the FRAX group than in the control group (13 versus 1%, P < 0.001). When embryos were transferred, ongoing pregnancy/live birth rates per transfer were similar (29 versus 36%, P = 0.54). CONCLUSIONS: Ovarian dysfunction in FRAX carriers is more prevalent and profound than previously appreciated, with a high cancelation rate and reduced efficiency of PGD. The main determinant for successful PGD for FRAX is ovarian dysfunction. When embryo transfer is possible, the results are comparable to PGD for other monogenic diseases.

Does estrogen directly modulate anti-müllerian hormone secretion in women?

OBJECTIVE: To investigate whether estrogen may modulate anti-müllerian hormone (AMH) expression in women. DESIGN: Prospective analysis. SETTING: Fertility clinic of tertiary university hospital. PATIENT(S): Cycling infertile women. INTERVENTION(S): Blood samples were taken at the early, middle, and late follicular phase in five groups: spontaneous cycle (n=10), ovulation induction with clomiphene-citrate (n=15) or gonadotropins (n=9), controlled ovarian hyperstimulation for IVF (COH-IVF; n=10) and in women who were treated with exogenous E2 for frozen-thawed embryo-transfer (FET) with no follicular development (n=20). MAIN OUTCOME MEASURE(S): AMH and E2 serum levels. RESULT(S): Basal serum AMH and E2 levels were similar in all groups. AMH levels were stable in all women during the follicular phase except for significant reduction in the COH-IVF group. In women in the FET group with high E2 levels, comparable to the COH-IVF group, AMH levels remained stable. CONCLUSION(S): In women, estrogen does not appear to have a direct role in AMH regulation.

Relationships between FSH, inhibin B, anti-Mullerian hormone, and testosterone during long-term treatment with the GnRH-agonist histrelin in patients with prostate cancer.

OBJECTIVES: Medical castration with long-acting GnRH-agonist (GnRHa) is a well-established treatment for metastatic prostate cancer. Our aim was to explore the relationships between FSH, inhibin B, anti-Mullerian hormone (AMH), and testosterone during treatment with an implant releasing GnRHa. DESIGN: Analysis of hormone levels in frozen serum samples. METHODS: Ten patients aged 77+/-7 (means+/-S.E.M.) years with prostate cancer were treated with the GnRHa histrelin for at least a year. Two weeks prior to insertion and for 3-4 months following removal the patients were treated with the antiandrogen flutamide. Serum inhibin B, FSH, testosterone, and AMH levels were measured retrospectively. RESULTS: FSH, inhibin B, and testosterone increased during antiandrogen administration and levels fell after implant insertion. Four weeks post insertion, FSH gradually increased while inhibin B and testosterone remained fully suppressed. AMH levels did not change during antiandrogen treatment, but increased following implant insertion and remained elevated for the duration of implant use. Following removal, FSH and testosterone increased, inhibin B remained low, while AMH decreased. CONCLUSIONS: The secondary increase in FSH following initial suppression with the implant is probably related to impaired inhibin B secretion. The lack of inhibin B response to the secondary increase in FSH suggests that long-term exposure of Sertoli-cells to GnRHa impairs their function. This effect appears to be selective since unlike inhibin B, AMH increased. In the absence of testosterone, FSH has a role in AMH regulation.

Ovarian hemorrhage after transvaginal ultrasonographically guided oocyte aspiration: a potentially catastrophic and not so rare complication among lean patients with polycystic ovary syndrome.

OBJECTIVE: To report the first case series of ovarian hemorrhage after transvaginal ultrasonographically guided oocyte aspiration (TVOA). DESIGN: Retrospective analysis. SETTING: In vitro fertilization unit of a tertiary university hospital. PATIENT(S): Patients who underwent TVOA during a 6-year period. INTERVENTION(S): Surgical intervention due to active bleeding from the ovary. MAIN OUTCOME MEASURE(S): Prevalence and risk factors. RESULT(S): Among 3,241 patients undergoing TVOA, 7 were diagnosed as having ovarian hemorrhage afterward. All patients were thin, with a body mass index of 19-21 kg/m(2), and 4 had polycystic ovary syndrome (PCOS). The prevalence of ovarian bleeding among lean patients with PCOS was 4.5%. The odds ratio for bleeding in lean patients with PCOS vs. all other patients was 50 (95% confidence interval 11-250). The interval between the TVOA and surgical intervention ranged from 5 to 18 hours (mean +/- SD, 11.4 +/- 5 hours). The Delta decrease in hemoglobin levels was 3.2-9 g/dL (mean 6.1 +/- 1.8). In 6 of the 7 patients, laparoscopically guided electrocoagulation was sufficient to achieve hemorrhagic control. CONCLUSION(S): Although acute hemorrhage is a rare event after TVOA, lean patients with PCOS specifically are at much higher risk for this complication.

What should be the first-line treatment for unexplained infertility in women over 40 years of age - ovulation induction and IUI, or IVF?

The tendency to postpone childbearing in developed countries and the relatively high rate of infertility in older women contribute to an increase in the portion of women aged 40 years and older opting for infertility treatments. The main factor for infertility in this group is oocyte senescence, but since this process does not have a specific diagnosis many of those will be classified as having 'unexplained infertility'. The efficacy of the traditional clinical approach for 'unexplained infertility' in older women is questionable. Reviewing the current literature, clomiphene citrate seems to be inefficient in this group of patients, while delivery rates of gonadotrophins and intrauterine insemination cycles are less than 5%. Although low in absolute terms, IVF is more efficient. The chance for delivery, however, diminishes with each year of age above 40. Therefore, after a short trial of gonadotrophins and intrauterine insemination, women aged 40-41 years should be quickly referred to IVF. At an older age, IVF is the primary treatment option.

Non-invasive prenatal diagnosis using cell-free fetal DNA in maternal plasma from PGD pregnancies.

Preimplantation genetic diagnosis (PGD) is usually used to establish a non-affected pregnancy for those couples facing a genetic risk of having an affected child. However, an invasive test is still recommended to all PGD patients due to the risk of misdiagnosis. The discovery of cell-free fetal DNA in maternal plasma provides the possibility for noninvasive prenatal diagnosis. Studies have shown that fetal single-gene disorders can be detected in cell-free fetal DNA by the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) assay with single-allele base extension reaction (SABER) approach or by the size-fractionation approach, whereby cell-free fetal DNA is enriched on the basis of its smaller size compared with maternal DNA fragments. Recent studies have indicated that a combination of the two approaches increases the accuracy of detection. This study combined the two methods and examined fetal paternally inherited gene mutations in maternal plasma obtained from four PGD-conducted pregnancies. The presence or absence of mutations was correctly detected in all cases. This combined method could be used for risk-free prenatal diagnosis of diseases caused by single-gene mutations, and in particular for couples who undergo PGD who opt not to perform invasive prenatal confirmation due to the risk of abortion.