Messenger RNA and protein expression of tumor necrosis factor α and its receptors in human follicular granulosa cells.

To evaluate the concentration of tumor necrosis factor α (TNF-α) and its soluble receptors (sTNFR I and II) in serum and follicular fluid (FF) at the time of oocyte retrieval and to detect expression of TNF-α and its receptors by luteinized granulosa cells (GCs). In a cross-sectional study and through an in vitro fertilization-intracytoplasmic sperm injection (IVF-ICSI) program, 81 women undergoing oocyte retrieval were recruited. Serum and FF were obtained from 81 women. GCs were pooled from 20 patients (from six different days of oocyte retrievals, 5-16 follicles per patient). TNF-α and its soluble receptors concentration were determined by enzyme-linked immunosorbent assay and also their expression by immune cytochemistry and reverse-transcription polymerase chain reaction analysis. The median TNF-α concentration in serum was 4.06 pg/ml (interquartile range [IQR], 3.71-6.14) and significantly higher than that in FF with 3.50 pg/ml (IQR, 3.05-5.01), p < 0.001. The sTNFR I and II levels in serum were lower and higher than FF, respectively. The TNF-α levels in serum and FF of good responders were higher than low responders (p = 0.017 and 0.021, respectively). TNF-α cut-off level for low responders versus good responders was 4.174 pg/ml in serum with a pregnancy rate of 25.8% and 40% for below and above of this level, respectively (p = 0.19). For FF, the cut-off value was 3.89 pg/ml. TNF-α and its receptors were expressed by GCs. The presence of TNF-α and its soluble receptors in serum and FF and their expression by GCs suggest an important role for this cytokine in ovarian function.

Preimplantation genetic testing for aneuploidy by microarray analysis of polar bodies in advanced maternal age: a randomized clinical trial.

STUDY QUESTION: Does preimplantation genetic testing for aneuploidy (PGT-A) by comprehensive chromosome screening (CCS) of the first and second polar body to select embryos for transfer increase the likelihood of a live birth within 1 year in advanced maternal age women aged 36-40 years planning an ICSI cycle, compared to ICSI without chromosome analysis? SUMMARY ANSWER: PGT-A by CCS in the first and second polar body to select euploid embryos for transfer does not substantially increase the live birth rate in women aged 36-40 years. WHAT IS KNOWN ALREADY: PGT-A has been used widely to select embryos for transfer in ICSI treatment, with the aim of improving treatment effectiveness. Whether PGT-A improves ICSI outcomes and is beneficial to the patients has remained controversial. STUDY DESIGN, SIZE, DURATION: This is a multinational, multicentre, pragmatic, randomized clinical trial with intention-to-treat analysis. Of 396 women enroled between June 2012 and December 2016, 205 were allocated to CCS of the first and second polar body (study group) as part of their ICSI treatment cycle and 191 were allocated to ICSI treatment without chromosome screening (control group). Block randomization was performed stratified for centre and age group. Participants and clinicians were blinded at the time of enrolment until the day after intervention. PARTICIPANTS/MATERIALS, SETTING, METHODS: Infertile couples in which the female partner was 36-40 years old and who were scheduled to undergo ICSI treatment were eligible. In those assigned to PGT-A, array comparative genomic hybridization (aCGH) analysis of the first and second polar bodies of the fertilized oocytes was performed using the 24sure array of Illumina. If in the first treatment cycle all oocytes were aneuploid, a second treatment with PB array CGH was offered. Participants in the control arm were planned for ICSI without PGT-A. Main exclusion criteria were three or more previous unsuccessful IVF or ICSI cycles, three or more clinical miscarriages, poor response or low ovarian reserve. The primary outcome was the cumulative live birth rate after fresh or frozen embryo transfer recorded over 1 year after the start of the intervention. MAIN RESULTS AND THE ROLE OF CHANCE: Of the 205 participants in the chromosome screening group, 50 (24%) had a live birth with intervention within 1 year, compared to 45 of the 191 in the group without intervention (24%), a difference of 0.83% (95% CI: -7.60 to 9.18%). There were significantly fewer participants in the chromosome screening group with a transfer (relative risk (RR) = 0.81; 95% CI: 0.74-0.89) and fewer with a miscarriage (RR = 0.48; 95% CI: 0.26-0.90). LIMITATIONS, REASONS FOR CAUTION: The targeted sample size was not reached because of suboptimal recruitment; however, the included sample allowed a 90% power to detect the targeted increase. Cumulative outcome data were limited to 1 year. Only 11 patients out of 32 with exclusively aneuploid results underwent a second treatment cycle in the chromosome screening group. WIDER IMPLICATIONS OF THE FINDINGS: The observation that the similarity in birth rates was achieved with fewer transfers, less cryopreservation and fewer miscarriages points to a clinical benefit of PGT-A, and this form of embryo selection may, therefore, be considered to minimize the number of interventions while producing comparable outcomes. Whether these benefits outweigh drawbacks such as the cost for the patient, the higher workload for the IVF lab and the potential effect on the children born after prolonged culture and/or cryopreservation remains to be shown. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the European Society of Human Reproduction and Embryology. Illumina provided microarrays and other consumables necessary for aCGH testing of polar bodies. M.B.'s institution (UZBrussel) has received educational grants from IBSA, Ferring, Organon, Schering-Plough, Merck and Merck Belgium. M.B. has received consultancy and speakers' fees from Organon, Serono Symposia and Merck. G.G. has received personal fees and non-financial support from MSD, Ferring, Merck-Serono, Finox, TEVA, IBSA, Glycotope, Abbott and Gedeon-Richter as well as personal fees from VitroLife, NMC Healthcare, ReprodWissen, BioSilu and ZIVA. W.V., C.S., P.M.B., V.G., G.A., M.D., T.E.G., L.G., G.Ka., G.Ko., J.L., M.C.M., M.P., A.S., M.T., K.V., J.G. and K.S. declare no conflict of interest. TRIAL REGISTRATION NUMBER: NCT01532284. TRIAL REGISTRATION DATE: 7 February 2012. DATE OF FIRST PATIENT'S ENROLMENT: 25 June 2012.

Correction to: Two-year outcome after recurrent implantation failure: prognostic factors and additional interventions.

The article Two-year outcome after recurrent implantation failure: prognostic factors and additional interventions.

Reduced ovarian reserve in young early breast cancer patients: preliminary data from a prospective cohort trial.

BACKGROUND: The numerous side effects of chemotherapy in patients with breast cancer are well known. However, the precise effects of chemotherapy on ovarian function in premenopausal women are poorly investigated. The patients are at risk of developing sexual hormone deficiency and impaired fertility. This prospective cohort study addresses predictive parameters of ovarian reserve after chemotherapy. METHODS: Fifty-one premenopausal women (28-46 years) with primary breast cancer were included in the trial. All of them received anthracycline-based chemotherapy (n = 18), or combinations with taxanes (n = 30), or anthracycline-free chemotherapy (n = 3). Changes in hormone levels (LH, FSH, E2 and Anti-Müllerian hormone (AMH)), antral follicle count (AFC), and amenorrhea were determined before (V1), and 6, 12 and 24 months after the initiation of chemotherapy (V2-V4). Quality of life parameters were evaluated. The additional impact of parity, BMI, and smoking on ovarian reserve was also assessed. RESULTS: AFC and AMH fell very markedly after chemotherapy and did not return to pre-treatment levels until V4. A significant positive correlation was noted in AFC before and 1 year after chemotherapy. AMH levels at V2-V4 were significantly correlated with those registered at V1. AFC and AMH were negatively correlated with age. Continued smoking had a significant detrimental effect on AFC after 24 months. LH and FSH levels increased between V1 and V2 and fell at V3 and V4, but stayed above pre-chemotherapy values. Two years after the start of chemotherapy 31/51 patients were amenorrhoic while 17 resumed their menstrual cycle; this was not influenced by the type of chemotherapy or age. Non-smokers were 13 times more likely to resume their menstruation than smokers. Quality of life (QL) was significantly lower 6 months after the initiation of chemotherapy. QL at one and 2 years after chemotherapy did not differ significantly from pre-chemotherapy scores. CONCLUSIONS: Our study contributes to a better understanding and prediction of ovarian reserve in young early breast cancer patients undergoing chemotherapy. The data suggest that personal counseling in regard of the preservation of fertility should be offered especially to patients of a higher age, with low AMH levels or low follicle counts. Patients should be advised to stop smoking in order to enhance the likelihood of preserving their fertility.

Oocyte morphology on day 0 correlates with aneuploidy as detected by polar body biopsy and FISH.

PURPOSE: For better selection of oocytes and embryos, preimplantation genetic screening (PGS) was introduced. As from the beginning of IVF, morphology was used as selection criteria; we investigated the combination of both. If there was a correlation between phenotype and genotype, invasive PGS might be replaced. METHOD: Therefore, 104 cycles with PGS were done by biopsy of the first polar body and FISH with five chromosomes. Morphology of the oocyte was recorded digitally and noted for 12 categories in 4-13 values; evaluation of the chromosomes was noted for five chromosomes in five values. Morphology and genetics were correlated to each other. RESULT: Correlations between morphology and genetics for day 0 were found: oocytes with an irregular or dark zona are less probable to have a normal chromosome 13 (80 vs. 53 %, p = 0.001). A medium amount of detritus in the perivitelline space makes it more probable to have a normal chromosome 18 (94 vs. 78 %, p = 0.001). A halo in the cytoplasm makes it less probable to be euploid for chromosome 22 (56 vs. 75 %, p = 0.018). For day 1, pattern "1, 2, 3 and fine" in the pronuclei makes it more probable to be euploid for chromosome 22 (78 vs. 63 %, p = 0.002). CONCLUSION: There are correlations between the oocyte genome and its morphology also on day 0. These correlations are not sufficient to replace PGS.

Sham-controlled implantation after preimplantation genetic screening by polar body biopsy and FISH.

PURPOSE: Preimplantation genetic screening wants to improve artificial reproductive technologies, primarily by raising the rates of pregnancy, implantation and birth. We investigated if embryos derived from oocytes detected euploid for five chromosomes implant better than those which were biopsied but where the genetic detection failed. They were nevertheless transferred, thus serving as a sham control. METHOD: From 2004 to 2008 we performed 104 cycles of PGS with laser biopsy of the first polar body and FISH with five chromosomes. It was offered to all patients with eight or more oocytes, free of charge. The average female age was 36 years. If no euploid oocytes were available, not detected oocytes were transferred. RESULT: In 104 cycles 99 embryo transfers (95 %) were performed, resulting in 28 pregnancies (27 %), 20 births (71 %) and 8 miscarriages (29 %). The implantation rate in the euploid group was 19 vs. 13 % in the not detected group (n.s.). This trend was the same independent of age and embryo morphology. CONCLUSION: The pregnancy rate does not differ significantly from the national average. The trend in better implantation rates of euploid oocytes justifies a continuation of studies in this matter.

Expression of interleukin-1ß and its receptor in human granulosa cells and their association with steroidogenesis.

This study aimed to investigate whether interleukin 1ß (IL-1ß) and soluble IL-1 receptor 2 (sIL-1R2) are expressed in human granulosa cells (GCs) and relate to ovarian steroidogenesis. Ninety-six women undergoing in vitro fertilization (IVF) were recruited. RT-PCR and immunocytochemistry were used to detect mRNAs and proteins of IL-1ß and IL-1R2, respectively. The steroidogenesis of primary cultured GCs was evaluated following treatment with either IL-1ß alone or IL-1ß and FSH in combination. There were positive correlations between serum IL-1ß and serum progesterone (r = 0.220, p = 0.032) and follicular fluid (FF) estradiol (r = 0.242, p = 0.018). Additionally, serum and FF sIL-1R2 were negatively and positively correlated with FF estradiol (r = -0.376, p = 0.005) and FF progesterone (r = 0.434, p = 0.001), respectively. The mRNA and protein expression of IL-1ß and IL-1R2 became evident in GCs. IL-1ß alone significantly increased estradiol secretion from GCs, but in the presence of FSH, it could notably promote progesterone secretion in addition to estradiol. In conclusion, IL-1ß and sIL-1R2 are expressed in human GCs and substantially contribute to ovarian steroidogenesis, suggesting that the IL-1ß system may be a potential target for optimizing ovarian hyperstimulation and steroidogenesis in IVF cycles.

Incidence of testicular malignancies and correlation to risk factors in a TESE population of subfertile men.

INTRODUCTION: In recent years, researchers have postulated a decreasing fertility potential of males and a rising incidence of testicular malignancies. MATERIALS AND METHODS: In this retrospective observational diagnostic multicenter study, 302 patient files of subfertile men whose testes were biopsied for TESE procedure were analysed. All patients referred to reproductive medicine centres in Northern Germany and they were identified by the cycle data collected by the German IVF register. A total of 280 patients (436 cycles) treated for intracytoplasmatic sperm injection after TESE procedures were eligible to be analysed. RESULTS: Our findings: 13.0% overall paternity rate before TESE procedure, 45.9% smokers, maldescensus testis was found on occasion (12.9%), and mumps orchitis previously occurred to 10 patients (3.6%). The tumour incidence rate at the time of testicle biopsy was 1.81% (= 5 pts.). Two of these patients had an anamnesis for maldescensus testis and one patient acquired mumps orchitis in childhood. CONCLUSION: Our data even reflects that tumour patients express an interest in having children after completion of cancer treatment, presenting four patients who had testicular biopsies after a previous malignancy. Moreover, there is evidence suggesting that environmental factors are causative for the trends in occurrence of male reproductive health problems. Within our highly selected population, the testicular tumour incidence rate is 100-fold higher than in a standard population. Supposing that the incidence rate of testicular malignancies among infertile men continues to increase in comparison to the incidence rate of the general male population, one has to count on an incremental number of males suffering from subfertility and testicular tumours.

Polar body array CGH for prediction of the status of the corresponding oocyte. Part I: clinical results.

BACKGROUND: Several randomized controlled trials have not shown a benefit from preimplantation genetic screening (PGS) biopsy of cleavage-stage embryos and assessment of up to 10 chromosomes for aneuploidy. Therefore, a proof-of-principle study was planned to determine the reliability of alternative form of PGS, i.e. PGS by polar body (PB) biopsy, with whole genome amplification and microarray-based comparative genomic hybridization (array CGH) analysis. METHODS: In two centres, all mature metaphase II oocytes from patients who consented to the study were fertilized by ICSI. The first and second PBs (PB1and PB2) were biopsied and analysed separately for chromosome copy number by array CGH. If either or both of the PBs were found to be aneuploid, the corresponding zygote was then also processed by array CGH for concordance analysis. RESULTS: Both PBs were biopsied from a total of 226 zygotes from 42 cycles (average 5.5 per cycle; range 1-15) in 41 couples with an average maternal age of 40.0 years. Of these, the ploidy status of the zygote could be predicted in 195 (86%): 55 were euploid (28%) and 140 were aneuploid (72%). With only one exception, there was at least one predicted aneuploid zygote in each cycle and in 19 out of 42 cycles (45%), all zygotes were predicted to be aneuploid. Fresh embryos were transferred in the remaining 23 cycles (55%), and one frozen transfer was done. Eight patients had a clinical pregnancy of which seven were evolutive (ongoing pregnancy rates: 17% per cycle and 30% per transfer). The ploidy status of 156 zygotes was successfully analysed by array CGH: 38 (24%) were euploid and 118 (76%) were aneuploid. In 138 cases complete information was available on both PBs and the corresponding zygotes. In 130 (94%), the ploidy status of the zygote was concordant with the ploidy status of the PBs and in 8 (6%), the results were discordant. CONCLUSIONS: This proof-of-principle study indicates that the ploidy of the zygote can be predicted with acceptable accuracy by array CGH analysis of both PBs.

Polar body array CGH for prediction of the status of the corresponding oocyte. Part II: technical aspects.

BACKGROUND: The purpose of this study was to assess the technical aspects related to polar body (PB) biopsy, which might have an influence on the results of the microarray comparative genomic hybridization analysis. Furthermore, a comparison was made between two biopsy methods (mechanical and laser). METHODS: Biopsy of the first and second PB (PB1 and PB2) was performed by mechanical- or laser-assisted biopsy in two different IVF centres. PBs were separately amplified by whole genome amplification. RESULTS: The method of biopsy, mechanical or laser had no influence on the proportion of successfully biopsied oocytes. Especially, for the PB2, the timing of biopsy after ICSI was directly correlated to amplification efficiency. CONCLUSIONS: Special care has to be taken with respect to the timing of biopsy of the PB2. Mechanical- and laser-assisted biopsy give the same performance in terms of diagnostic efficiency.

Circulating concentration of stem cell factor in serum of stimulated IVF patients.

Stem cell factor (SCF) plays a major role in haematopoiesis and spermatogenesis, and possibly female fertility. This study investigated the role of changes in SCF concentrations in 74 assisted conception patients. In group 1 (n=74) SCF concentration was assessed in serum and follicular fluid (FF) on the day of follicular puncture (FP) and compared in serum and FF in response to ovarian stimulation between low (n=25), moderate (n=26) and high (n=14) responders. In group 2 (n=30) serum for SCF assessment was collected throughout the menstrual cycle until gestation. SCF concentration related to the number of follicles in serum and in FF decreased from low to moderate and high responders (P<0.001); pregnancy rates were 20.0%, 34.6% and 50.1%, respectively (P=0.05). SCF in serum increased from stimulation days 6-8 to 9-11 and peaked on the day of human chorionic gonadotrophin injection (P=0.03). The SCF concentrations dropped slightly on the day of FP, increased significantly to the day of pregnancy confirmation and reached highest concentration (P=0.02) during gestation. SCF is involved in follicle development and may be a predictor of IVF outcome.

What next for preimplantation genetic screening? A polar body approach!

Screening of human preimplantation embryos for numerical chromosome abnormalities has been conducted mostly at the preimplantation stage using fluorescence in situ hybridization. However, it is clear that preimplantation genetic screening (PGS) as it is currently practiced does not improve live birth rates. Therefore the ESHRE PGS Task Force has decided to start a proof of principle study with the aim of determining whether biopsy of the first and second polar body followed by subsequent analysis of the complete chromosome complement of these polar bodies using an array based technique enables a timely identification of the chromosomal status of an oocyte. If the principle of this approach can be proven, it is obvious that a multicentre randomized controlled trial should then be started to determine the clinical value of this technique. In this way the ESHRE PGS Task Force hopes to redirect preimplantation screening from the blind alley to the main road of assisted reproduction.

Theory and practice of preimplantation genetic screening (PGS).

OBJECTIVE: In the context of artificial reproductive technology (ART) treatments with in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), the purpose of genetic screening of oocytes and embryos in vitro prior to implantation (preimplantation genetic screening, PGS) is highly controversial. Therefore, an analysis of the following theoretical prerequisites is presented: the abstract investigation method and the medical diagnostic decision, indication and ethical acceptability. The first is a scientific task, while the other is a physician's task. THEORY OF PGS: As the new term preimplantation genetic diagnosis for aneuploidies (PGT-A) does not sufficiently take into account probable future developments, PGS is retained here. In clinical practice, PGS refers to the biopsy of polar bodies, blastomeres or trophoblast cells with indication-dependent genetic analysis. Goals include increasing pregnancy rates and reducing abortion rates, multiple birth rates, malformation rates and pointless ART treatments. To improve the pregnancy rate, PGS makes no sense if a stochastic selection advantage is not to be expected. Patients may have to choose between the chance of rapid success with a first fresh embryo transfer of blastocysts and a possibly higher overall cumulative chance of pregnancy from fresh and thawed transfers of four-to eight-cell embryos. It is neither necessary nor useful to make every medical decision dependent on randomized controlled trials (RCTs). The randomization of patients is not indicated if observational studies have not shown a positive result. For a "proof-of-principle study", the numerator and denominator of the cascade of parameters for success must be close to each other and far apart in an "efficacy study". The randomization may only be performed before the biopsy. PRACTICE: Following the introduction of blastocyst biopsy and comprehensive chromosome screening (CCS) with, for example, aCGH and NGS, referred to as "PGS 2.0″, all RCTs since 2012 have found a positive effect. DISCUSSION: There is still disagreement about the interpretation of the results of PGS 2.0, but the overwhelming view in opinion publications seems to be that it works. This fits with the increasing global commitment to PGS 2.0. CONCLUSION: PGS may be beneficial if used with strict indications, taking into account stochastics and the will of the patient. The task of the physician, similar to counselling in prenatal medicine, is as follows: present all methods of investigation and respect the will of the patient.

Provision and quality assurance of preimplantation genetic diagnosis in Europe.

Preimplantation genetic diagnosis (PGD) is now well established and provided in many European countries. However, regulations, professional standards and accreditation requirements can differ notably. Furthermore, no comprehensive independent data exist either about practice and provision in Europe or about the quality assurance practices and procedures designed to optimize the quality of the results. Consequently, a study was launched to obtain knowledge, currently lacking, of the provision and quality assurance of PGD services and cross-border activities in Europe. An online questionnaire was developed and sent to PGD providers, and expert opinions were obtained through interviews with professionals in specific countries. Information was gathered from 53 centres offering PGD in 17 European countries. There is a diverse array of tests available, with a trend for custom-made services. Although half of the centres have a designated quality manager, just 33% have achieved or are preparing for accreditation or certification. About 66% of the centres responded that they did not participate in external quality assessment, a problem exacerbated by the lack of existing PGD-specific schemes. Approximately 19% of the centres do not keep data on accuracy and 9% do not even follow up until birth. PGD is an expanding activity with an increasing international flow that accounts for approximately one-third of the activity reported. The survey highlights a significant need for improvement in quality assurance in PGD centres. On the positive side, important improvements in the quality management of these services are expected with the European Tissue Directive entering into force.

The Role of Interleukin-18 in Serum and Follicular Fluid during In Vitro Fertilization and Intracytoplasmic Sperm Injection.

Cytokines are key modulators of the immune system and play an important role in the ovarian cycle. IL-18 levels in serum and follicular fluid were analyzed in women undergoing in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) treatment. The cohort study group consisted of 90 women, who were undergoing IVF or ICSI. The body mass index (BMI) was determined in all patients; IL-18 levels were measured in follicular fluid and serum. IL-18 levels in serum were significantly higher than those in follicular fluid. The median level in serum was 162.75 (80.21) pg/mL and that in follicular fluid, 138.24 (91.78) pg/mL. Women undergoing IVF treatment had lower IL-18 levels in serum (median, 151.19 (90.73) pg/mL) than those treated with ICSI (median, 163.57 (89.97) pg/mL). The correlation between IL-18 levels in serum and BMI was statistically significant, as well as the correlation between IL-18 levels in follicular fluid and ovarian stimulation response (p = 0.003). IL-18 was correlated with the response to ovarian stimulation and was the reason for successful pregnancy after IVF or ICSI treatment. Among other cytokines, IL-18 appears to be a promising prognostic marker of success in reproductive treatment and should be evaluated as such in further prospective studies.

Expression of mRNA and protein of macrophage colony-stimulating factor and its receptor in human follicular luteinized granulosa cells.

OBJECTIVE: To evaluate the concentration of macrophage colony-stimulating factor (M-CSF) in serum and follicular fluid (FF) at the time of oocyte retrieval and to detect expression of M-CSF and its receptor by luteinized granulosa cells (GCs). DESIGN: Collection of serum and FF at the time of oocyte retrieval. SETTING: A university IVF- intracytoplasmic sperm injection (ICSI) program. PATIENT(S): Serum and FF were obtained from 85 women undergoing oocyte retrieval. INTERVENTION(S): Serum and FF were obtained from 85 women. The GCs were pooled from 15 (3 x 5) patients (3-14 oocytes each). MAIN OUTCOME MEASURE(S): The M-CSF concentration was determined by ELISA, the expression of M-CSF and its receptor by the immunocytochemical technique and reverse transcription polymerase chain reaction analysis. In addition, M-CSF expression was investigated by cell culture time course studies. RESULTS: The median M-CSF concentration in FF (2,409.2 pg/mL) was significantly higher than that in serum (242.5 pg/mL). The M-CSF and its receptor were expressed by GCs. CONCLUSION(S): The significantly higher level of M-CSF in FF than in serum and the expression of M-CSF and its receptor in FF by GCs suggest an important role for this growth factor in ovarian function.

Cut-Off Levels of Anti-Mullerian Hormone for The Prediction of Ovarian Response, In Vitro Fertilization Outcome and Ovarian Hyperstimulation Syndrome.

BACKGROUND: Evaluation of anti-mullerian hormone (AMH) cut-off levels in as- sisted reproductive technology (ART) as predictive factor for individualization of stimulation protocols and to avoid ovarian hyperstimulation syndrome (OHSS). MATERIALS AND METHODS: In a retrospective study, 177 infertile patients were as- sessed for AMH in serum and follicular fluid (FF) on the day of follicular puncture (FP), between 2012 and 2013 in Kiel, Germany. AMH levels and pregnancy rates were compared between low, moderate and high responders and cut-off levels of low and high responders. AMH cut-off levels in pathological cases were evaluated in analysis 1 (OHSS) and in analysis 2 [polycystic ovarian syndrome, (PCOS)] and compared in analysis 3 to normal endocrinological parameters. RESULTS: AMH levels in FF were higher than in serum (P<0.001). AMH levels in serum and FF increased from low through moderate to high responders (P<0.001). Pregnancy rates were 14.7, 23.3 and 44.9% (P=0.009), respectively. AMH cut-off level for poor responders was 0.61 ng/ml in serum with a pregnancy rate of 13.8 and 37.1% for below and above of this level, respectively. For FF, it was 1.43 ng/ml. AMH levels in analysis 1 and 2 were significantly higher than in analysis 3 (P=0.001). AMH cut-off level for OHSS was 1.5 ng/ml in serum with OHSS rates of 80.8 and 19.2 % for above and below of the level, respectively. For FF, it was 2.7 ng/ml. PCOS patients had an AMH cut-off level of 3.9 ng/ml in serum and 6.8 ng/ml in FF, resulting in a PCOS rate of 100% above this level. CONCLUSION: AMH levels can help to assess ovarian response potential and guide ovarian stimulation while avoiding OHSS.

Detection of granulocyte colony-stimulating factor and its receptor in human follicular luteinized granulosa cells.

OBJECTIVE: To evaluate concentration of granulocyte colony-stimulating factor (G-CSF) in serum and follicular fluid (FF) at the time of oocyte retrieval and to detect expression of G-CSF and its receptor by luteinized granulosa cells (GCs). DESIGN: Collection of serum and FF at the time of oocyte retrieval. SETTING: A university IVF-ICSI program. PATIENT(S): Serum and FF were obtained from 82 women undergoing oocyte retrieval. INTERVENTION(S): Serum and FF were obtained from 82 women. Granulosa cells were pooled from 15 patients (three experiments with five patients each; 3-14 oocytes each). MAIN OUTCOME MEASURE(S): Granulocyte colony-stimulating factor concentration was determined by ELISA, the expression of G-CSF, and its receptor by the immunocytochemical technique and reverse transcriptase polymerase chain reaction analysis. Additionally, G-CSF expression was investigated by cell culture time course studies. RESULT(S): The median G-CSF level in FF (117.98 pg/mL) was significantly higher than that in serum (67.5 pg/mL). Granulocyte colony-stimulating factor and its receptor were expressed by GCs. CONCLUSION(S): The significantly higher level of G-CSF in FF than in serum and the expression of G-CSF and its receptor in FF by GCs suggest an important role for this growth factor in ovarian function.

Stress relief after infertility treatment--spontaneous conception, adoption and psychological counselling.

OBJECTIVE: In this study, we sought to evaluate characteristics of couples with spontaneous conceptions after treatment with assisted reproductive technologies (ART). STUDY DESIGN: Data from 254 couples who underwent 1127 therapy cycles between November 1987 and February 1997, were analyzed. Chi-Square (chi(2)) test and Student's t-test were used. P<0.05 was considered significant. RESULTS: Spontaneous pregnancies occurred in 14% of all treated couples. Psychological counselling only was performed in 21% but was observed significantly more frequently among patients without later spontaneous conception. Ten percent of all treated couples applied for adoption. The miscarriage rate was significantly higher in the group of treatment dependent pregnancies compared to the group of patients with later spontaneous conception (27% versus 9%). The spontaneous conception rate differed significantly depending on women's age and normal semen analysis. CONCLUSION: Appearance of spontaneous conception after ART-procedures should be taken into account in the first patient's interview. Depending on women's age and andrological parameters, treatment-success will differ. The positive impact of psychological counselling for stress relief during and after therapy should also be noted, even though a statistically significant impact could not be demonstrated in the present study. Adoption should be discussed as an alternative to overcome infertility.

Array-based DNA methylation profiling in male infertility reveals allele-specific DNA methylation in PIWIL1 and PIWIL2.

OBJECTIVE: To identify CpG sites differentially methylated in peripheral blood of men with idiopathic infertility due to impaired spermatogenesis as compared with fertile controls. DESIGN: DNA methylation profiling on peripheral blood samples using the HumanMethylation450 BeadChip (Illumina) in patients and controls, single-nucleotide polymorphism (SNP) typing by Sanger sequencing. SETTING: University institute in cooperation with genetic and infertility clinics. PATIENT(S): 30 infertile men with normal CFTR and AZF tests and karyotype, and 10 fertile male controls. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): DNA methylation levels at CpG sites. RESULT(S): We identified 471 CpGs (287 genes) as differentially methylated between patients and controls. These were significantly enriched for the gene ontology functions MHC class II receptor activity and piwi-interacting (piRNA) binding. The latter was associated with two methylation-sensitive SNPs in the genes PIWIL1 and PIWIL2, respectively, which showed significant allele distribution skewing in the infertile cohort. We found that 445 (94.5%) of 471 differentially methylated CpGs were associated with SNPs, but 26 (15 genes) were not genomically templated, including the ENO1, MTA2, BRSK2, and LBX2 genes previously associated with fertility and spermatogenesis. CONCLUSION(S): Our study identifies surrogate DNA methylation markers for idiopathic infertility in peripheral blood and suggests that allele-specific DNA methylation differences at regulatory sites of genes involved in piRNA regulation are associated with disturbed spermatogenesis.