Reproductive outcomes of women aged 40 and older undergoing IVF with donor sperm.

STUDY QUESTION: Do women ≥40 years old without a male partner who utilize donor sperm have the same reproductive outcomes as those who utilize their partner's sperm? SUMMARY ANSWER: After controlling for relevant confounders, women ≥40 years old using donor sperm for IVF have significantly higher odds of having a live birth compared to those utilizing their partner's sperm. WHAT IS KNOWN ALREADY: Women who are unpartnered or in same-sex relationships are by definition not infertile, but may choose to conceive using donor sperm. It is not known how IVF outcomes are affected with the use of donor sperm compared to women utilizing their partner's sperm, particularly at very advanced maternal ages. STUDY DESIGN, SIZE, DURATION: This is a retrospective cohort study conducted at a university-affiliated center of women undergoing IVF with fresh embryo transfer between 2008 and 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients were divided into two groups based on the ejaculated sperm source utilized: donor or partner sperm. Live birth rate was the primary outcome. Pregnancy rate was the secondary outcome. Multivariable logistic regression was performed and adjusted for age, the developmental stage of the embryo, and the number of embryos transferred. Unadjusted odds ratio (OR) and adjusted OR (aOR) with 95% CI for pregnancy and live birth were estimated. Statistical significance was denoted by P < 0.05. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 3910 cycles in women ≥40 years old were analyzed, of which 307 utilized donor sperm and 3603 utilized their partner's sperm to conceive. In the univariate analysis, patients utilizing donor sperm were found to have similar pregnancy rates as those utilizing partner sperm (41.0 vs 39.8%, OR: 0.95, 95% CI: 0.75-1.20). After adjusting for age, the number of embryos transferred and the developmental stage of the embryos, the model estimates did not vary (aOR: 1.22, 95% CI: 0.95-1.56). Similarly, the univariate analysis for live birth did not demonstrate a difference between groups (19.2 vs 17.8%, OR: 0.91, 95% CI: 0.67-1.22). However, after a similar adjustment was made for confounders, the use of donor sperm was associated with statistically significant increased odds of live birth (aOR: 1.38, 95% CI: 1.01-1.88). LIMITATIONS, REASONS FOR CAUTION: As with any retrospective study, the potential for residual confounding exists, despite attempts to control for this with regression modeling. WIDER IMPLICATIONS OF THE FINDINGS: Women ≥40 years old who are unpartnered or in same-sex relationships can be counseled that their odds of a live birth are slightly better than women in heterosexual relationships utilizing their partner's sperm. These findings serve to further refine and individualize counseling on the expected IVF outcomes for women in this population. STUDY FUNDING/COMPETING INTEREST(S): No funding was sought for this study. The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

No effect of ovarian stimulation and oocyte yield on euploidy and live birth rates: an analysis of 12 298 trophectoderm biopsies.

STUDY QUESTION: Does ovarian stimulation affect embryo euploidy rates or live birth rates (LBRs) after transfer of euploid embryos? SUMMARY ANSWER: Euploidy rates and LBRs after transfer of euploid embryos are not significantly influenced by gonadotropin dosage, duration of ovarian stimulation, estradiol level, follicle size at ovulation trigger or number of oocytes retrieved, regardless of a woman's age. WHAT IS KNOWN ALREADY: Aneuploidy rates increase steadily with age, reaching >80% in women >42 years old. The goal of ovarian stimulation is to overcome this high aneuploidy rate through the recruitment of several follicles, which increases the likelihood of obtaining a euploid embryo that results in a healthy conceptus. However, several studies have suggested that a high response to stimulation might be embryotoxic and/or increase aneuploidy rates by enhancing abnormal segregation of chromosomes during meiosis. Furthermore, a recent study demonstrated a remarkable difference in euploidy rates, ranging from 39.5 to 82.5%, among young oocyte donors in 42 fertility centres, potentially suggesting an iatrogenic etiology resulting from different stimulation methods. STUDY DESIGN, SIZE, DURATION: This is a retrospective cohort study that included 2230 in vitro fertilisation (IVF) with preimplantation genetic testing for aneuploidy (PGT-A) cycles and 930 frozen-thawed single euploid embryo transfer (FET) cycles, performed in our centre between 2013 and 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 12 298 embryos were analysed for ploidy status. Women were divided into five age groups (<35, 35-37, 38-40, 41-42 and >42 years old). Outcomes were compared between different durations of stimulation (<10, 10-12 and ≥13 days), total gonadotropin dosages (<4000, 4000-6000 and >6000 IU), numbers of oocytes retrieved (<10, 10-19 and ≥20 oocytes), peak estradiol levels (<2000, 2000-3000 and >3000 pg/mL), and sizes of the largest follicle on the day of trigger (<20 and ≥20 mm). MAIN RESULTS AND THE ROLE OF CHANCE: Within the same age group, both euploidy rates and LBRs were comparable between cycles regardless of their differences in total gonadotropin dosage, duration of stimulation, number of oocytes harvested, size of the largest follicles or peak estradiol levels. In the youngest group, (<35 years, n = 3469 embryos), euploidy rates were comparable between cycles with various total gonadotropin dosages (55.6% for <4000 IU, 52.9% for 4000-6000 IU and 62.3% for >6000 IU; P = 0.3), durations of stimulation (54.4% for <10 days, 55.2% for 10-12 days and 60.9% for >12 days; P = 0.2), number of oocytes harvested (59.4% for <10 oocytes, 55.2% for 10-19 oocytes and 53.4% for ≥20 oocytes; P = 0.2), peak estradiol levels (55.7% for E2 < 2000 pg/mL, 55.4% for E2 2000-3000 pg/mL and 54.8% for E2 > 3000 pg/mL; P = 0.9) and sizes of the largest follicle (55.6% for follicles <20 mm and 55.1% for follicles ≥20 mm; P = 0.8). Similarly, in the oldest group (>42 years, n = 1157 embryos), euploidy rates ranged from 8.7% for gonadotropins <4000 IU to 5.1% for gonadotropins >6000 IU (P = 0.3), from 10.8% for <10 days of stimulation to 8.5% for >12 days of stimulation (P = 0.3), from 7.3% for <10 oocytes to 7.4% for ≥20 oocytes (P = 0.4), from 8.8% for E2 < 2000 pg/mL to 7.5% for E2 > 3000 pg/mL (P = 0.8) and from 8.2% for the largest follicle <20 mm to 8.9% for ≥20 mm (P = 0.7). LBRs after single FET were also comparable between these groups. LIMITATIONS, REASONS FOR CAUTION: Although this large study (2230 IVF/PGT-A cycles, 12 298 embryos and 930 single FET cycles) demonstrates the safety of ovarian stimulation in terms of aneuploidy and implantation potential of euploid embryos, a multi-centre study may help to prove the generalisability of our single-centre data. WIDER IMPLICATIONS OF THE FINDINGS: These findings reassure providers and patients that gonadotropin dosage, duration of ovarian stimulation, estradiol level, follicle size at ovulation trigger and number of oocytes retrieved, within certain ranges, do not appear to significantly influence euploidy rates or LBRs, regardless of the woman's age. STUDY FUNDING/COMPETING INTEREST(S): No external funding was received and there are no competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.

A comparison of morphokinetic markers predicting blastocyst formation and implantation potential from two large clinical data sets.

PURPOSE: To demonstrate whether the standard morphokinetic markers used for embryo selection have a similar relationship to blastocyst formation and implantation in two large clinical data sets. METHODS: This is a retrospective cohort analysis striving to answer two distinct questions utilizing data sets from two large IVF clinics. Blastocysts (BL) and implanted blastocysts (I) in both clinics, IVI-Valencia (BL = 11,414, I = 479) and WMC (BL = 15,902; I = 337), were cultured in a time-lapse system (EmbryoScope, Vitrolife, Sweden). The study was designed to assess the relationship between early morphokinetic hallmarks and BL development, with a secondary analysis of implantation rates following single-embryo day 3 and day 5 transfers. RESULTS: We performed a detailed graphical analysis for t3, t5, duration of the second cell cycle (cc2) (t3-t2), and the ratio (t5-t3)/(t5-t2). The t5 timing was not affected between the clinics. However, Weill Cornell Medicine's (WCM) proportions were significantly affected by having BL vs. not. A significant decrease of blastocysts with longer t5 in WCM data, while t5 was more informative in the IVI data set for the implantation rate. CONCLUSIONS: Morphokinetic intervals for early cleavages were distributed differently between the clinics. Incorporation of embryo-selection algorithms depends on the individual clinic's selected developmental hallmarks, all of which must be validated before incorporation into clinical practice.

A treatment algorithm for couples with unexplained infertility based on sperm chromatin assessment.

OBJECTIVE: To design a reproductive treatment algorithm based on the sperm DNA fragmentation (SDF) for couples with unexplained infertility following a poor intrauterine insemination (IUI) outcome. DESIGN: Couples that failed IUI with no apparent reproductive issue in both partners were allocated to diverse reproductive treatments on the basis of SDF. SETTING: Reproductive medical center in an academic setting. PATIENT(S): Over 4 years, couples with an unexpected poor IUI outcome and no apparent female or male partner reproductive issues were recruited. INTERVENTION(S): IUI, IVF, and ICSI were performed in the standard fashion following sperm SDF assays. MAIN OUTCOMES MEASURE(S): Fertilization rate, implantation rate, pregnancy characteristics, and delivery rates. RESULT(S): A total of 354 couples with unexplained infertility and normal semen parameters underwent 1133 IUI cycles. Clinical pregnancy rate (CPR) with IUI at our center in an age-matched cohort is 23.9% while the study cohort had 1.8%. Following SDF assessment, couples with failed IUI attempts but normal SDF (SCSA 9.8 ± 4.6%; TUNEL 11.8 ± 6.2%) underwent IVF with a CPR of 12.7%; those with abnormal SDF underwent ICSI with ejaculated spermatozoa, resulting in a CPR of 18.7%. This group included couples with normal SDF that had failed IVF. Couples with abnormal SDF that failed ICSI with ejaculated spermatozoa achieved a CPR of 31.0% with surgically retrieved spermatozoa. CONCLUSION(S): Couples with unexplained infertility that present with unexpectedly poor IUI outcomes can be funneled into a treatment algorithm guided by the integrity of the sperm genome for higher chances of pregnancy using an alternate method of insemination.

Development of ICSI.

The first conception outside of the human body that led to the birth of Louise Brown was a tremendous accomplishment, which opened the door to the utilization of assisted reproductive techniques globally. This brought the understanding that accomplishing life in a dish required several steps, the most obvious being the timing and characteristics of fertilization. It soon became obvious in the 1980s that the most disappointing phenomenon was unexpected and complete fertilization failure. Among the approaches that were attempted to treat male factor infertility, ICSI surfaced as the technique that brought the ratio of the gametes to 1:1 and was also able to grant consistent fertilization and a higher pregnancy rate. ICSI has now been implemented for a quarter of a century, proving itself as the ultimate technique utilizing ejaculated spermatozoa independent of the semen parameters and is the sole insemination method to be used with surgically retrieved spermatozoa. There are currently various indications for ICSI that are widely adopted, rendering it the most popular insemination method worldwide. The reliability of ICSI ensures its employment in upcoming techniques involving in vitro spermatogenesis and neogametogenesis.

A rationale for biopsying embryos reaching the morula stage on Day 6 in women undergoing preimplantation genetic testing for aneuploidy.

STUDY QUESTION: Is there a benefit to assessing ploidy in delayed embryos reaching the morula stage on Day 6 of development? SUMMARY ANSWER: Day-6 morulae should be considered for biopsy in women <40 years old undergoing preimplantation genetic testing for aneuploidy (PGT-A) because they are associated with acceptable, albeit reduced, euploidy and implantation rates (IRs). WHAT IS KNOWN ALREADY: Embryo development and morphology have been shown to correlate with aneuploidy and pregnancy rates. During PGT-A cycles, embryos are biopsied if they reach the blastocyst stage by Day 5 or 6, whereas slow-developing embryos are typically deselected and discarded. Determining the viability of slow-developing embryos is particularly relevant for women undergoing PGT-A who have diminished ovarian reserve and a relatively low blastocyst yield. STUDY DESIGN, SIZE, DURATION: This is a retrospective cohort study that was performed at an academic medical center. Patients who underwent IVF with PGT-A were reviewed for inclusion. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 1615 cycles were reviewed. All cycles which involved a biopsy of a cavitating or compacted morula on Day 6 were included (n = 763). PGT-A was performed using array comparative genomic hybridization. The aneuploidy and implantation of morulae were compared to those of blastocysts originating from the same couples. MAIN RESULTS AND THE ROLE OF CHANCE: The study included 763 cycles in which 1260 morulae and 3014 blastocysts were biopsied. Women were divided into four age groups (<35, 35-37, 38-39 and ≥40 years): the prevalence of aneuploidy was consistently lower among blastocysts (40.3, 50.8, 56 and 78.3%, respectively) than among compacted morulae (68.7, 75.5, 88.9 and 98.1%, respectively) and cavitating morulae (57, 66.4, 81 and 91.6%, respectively) throughout the different age groups (P < 0.001). Of note, the majority of compacted morulae (98.1%) and cavitating morulae (91.6%) were aneuploid in women aged ≥40 years. Compacted and cavitating morulae had significantly higher rates of complex aneuploidy, which involves ≥3 chromosomes, compared with blastocysts (P < 0.001). Furthermore, euploid morulae were associated with a significantly lower IR (28.2 versus 54.6%; P = 0.002) and live birth rate (23.1 versus 55.0%; P = 0.001) compared to euploid blastocysts. LIMITATIONS REASONS FOR CAUTION: This study confirms that Day-6 morulae should not be discarded in young women undergoing PGT-A. However, a potential drawback of biopsying embryos at the morula stage is the inability to distinguish between inner cell mass and trophectoderm cell origin. The sample size of euploid morula transfer cycles in this study was limited. Thus, a larger cohort would be beneficial to validate the reassuring live birth and spontaneous abortion rates reported here. Furthermore, the reproducibility of our findings should be determined at different centers. WIDER IMPLICATIONS OF THE FINDINGS: Although Day-6 morulae are associated with higher aneuploidy rates and lower IRs compared to blastocysts, they still yielded successful pregnancies. Therefore, testing Day-6 morulae should be considered, especially for women <40 years old who are undergoing PGT-A with a small cohort of available blastocysts for biopsy. STUDY FUNDING/COMPETING INTEREST(S): The authors have nothing to disclose. They received no specific funding for this work. TRIAL REGISTRATION NUMBER: N/A.

Premature recruitment of oocyte pool and increased mTOR activity in Fmr1 knockout mice and reversal of phenotype with rapamycin.

While mutations in the fragile X mental retardation-1 (FMR1) gene are associated with varying reproductive outcomes in females, the effects of a complete lack of FMR1 expression are not known. Here, we studied the ovarian and reproductive phenotypes in an Fmr1 knockout (KO) mouse model and the role of mammalian target of rapamycin (mTOR) signaling. Breeding, histologic and mTOR signaling data were obtained at multiple time points in KO and wild type (WT) mice fed a control or rapamycin (mTOR inhibitor) diet. KO mice showed an earlier decline in ovarian reserve than WT mice with an increased proportion of activated follicles. mTOR and phosphorylated S6 kinase (p-S6K) levels, a measure of downstream mTOR signaling, were elevated in the KO ovaries. Rapamycin blocked these effects in KO mice, and increased the primordial follicle pool and age of last litter in WT mice. Our data demonstrates an early decline in reproductive capacity in Fmr1 KO mice and proposes that premature recruitment of the primordial pool via altered mTOR signaling may be the mechanism. Reversal of phenotypes and protein levels in rapamycin-treated KO mice, as well as increased reproductive lifespan of rapamycin-fed WT mice, suggest the mTOR pathway as a potential therapeutic target.

Intracytoplasmic sperm injection: state of the art in humans.

Among infertile couples, 25% involve both male and female factors, while male factor alone accounts for another 25% due to oligo-, astheno-, teratozoospermia, a combination of the three, or even a complete absence of sperm cells in the ejaculate and can lead to a poor prognosis even with the help of assisted reproductive technology (ART). Intracytoplasmic sperm injection (ICSI) has been with us now for a quarter of a century and in spite of the controversy generated since its inception, it remains in the forefront of the techniques utilized in ART. The development of ICSI in 1992 has drastically decreased the impact of male factor, resulting in millions of pregnancies worldwide for couples who, without ICSI, would have had little chance of having their own biological child. This review focuses on the state of the art of ICSI regarding utility of bioassays that evaluate male factor infertility beyond the standard semen analysis and describes the current application and advances in regard to ICSI, particularly the genetic and epigenetic characteristics of spermatozoa and their impact on reproductive outcome.

Supraphysiologic estradiol is an independent predictor of low birth weight in full-term singletons born after fresh embryo transfer.

STUDY QUESTION: Is supraphysiologic estradiol (E2) an independent predictor of low birth weight (LBW) in singletons born after fresh IVF-embryo transfer (ET) cycles? SUMMARY ANSWER: Our results suggest that E2 > 2500 pg/ml is an independent predictor for LBW in full-term singletons born to normal responder patients undergoing fresh IVF-ET cycles. WHAT IS KNOWN ALREADY: The pathogenesis of LBW in IVF singletons remains unknown. However, recent studies have suggested that the hyperestrogenic milieu generated during ovarian stimulation may create a sub-optimal peri-implantation environment, leading to placental dysfunction, and therefore, LBW. STUDY DESIGN, SIZE, DURATION: Retrospective cohort study of normal responder patients, <40 years old, undergoing fresh IVF-ET cycles resulting in live singleton births between January 2005 and June 2014. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 6419 patients had live births after fresh IVF-ET during the study period, of which 2348 (36.6%) patients were excluded due to multiple gestation, vanishing twins or incomplete records. Perinatal outcomes recorded for all patients included birth weight, gestational age (GA) at delivery, mode of delivery and gender. Term birth, preterm birth (PTB) and LBW incidence proportions were plotted against E2 level on the day of trigger. The term LBW incidence proportion (i.e. singletons born at GA ≥ 37 weeks with birth weight <2500 g) was considered the primary outcome of interest. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 4071 patients with live singleton births were included. The median age, BMI, E2 level and birth weight for the study cohort was 36 (33-39) years, 22.3 (20.4-25.0) kg/m2, 1554 (1112.7-2179) pg/ml and 3289 (2920-3628) g, respectively. The incidence proportion of LBW rose from 6.4% (E2 2001-2500 pg/ml) to 20.7% (E2 3501-4000 pg/ml), without a corresponding rise in the incidence proportion of PTB. The odds of term LBW with E2 > 2500 pg/ml were 6.1-7.9 times higher compared to the referent E2 group. Multivariable logistic regression analysis revealed that E2 was an independent predictor for term LBW, even after adjusting for age, BMI, race, parity, infertility diagnosis, duration of ovarian stimulation, gonadotropin dosage and method of insemination (adjusted odds ratio 10.8, 95% CI 9.2-12.5). Receiver operating characteristic analysis generated an AUC estimate of 0.85 for E2 level as a predictor of LBW. LIMITATIONS REASONS FOR CAUTION: The current study did not include analyses of hypertensive disorders of pregnancy or placental abnormalities. Furthermore, all patients were normal responders and of normal BMI, possibly limiting the overall generalizability of the study. Finally, as with any retrospective study, prospective data are required to validate the role of E2 in predicting LBW. WIDER IMPLICATIONS OF THE FINDINGS: Our results emphasize the importance of minimizing the supraphysiologic elevations of E2 levels during ovarian stimulation in fresh IVF-ET cycles. This, in turn, can optimize the early peri-implantation environment and mitigate adverse perinatal outcomes such as LBW. STUDY FUNDING/COMPETING INTEREST(S): Dr Paul J. Christos was partially supported by the following grant: Clinical and Translational Science Center at Weill Cornell Medical College (UL1-TR000457-06). TRIAL REGISTRATION NUMBER: N/A.

Thiazolidinediones (TZDs) affect osteoblast viability and biomarkers independently of the TZD effects on aromatase.

Thiazolidinediones (TZDs) are insulin sensitizers used for treatment of diabetes. We have previously reported that TZDs reduce estrogen synthesis by inhibiting aromatase activity in human granulosa cells (HGC). Multiple clinical trials demonstrated that TZDs increase the risk of fractures in postmenopausal women with type 2 diabetes. We studied mouse osteoblasts alone or in a co-culture with HGC to determine whether TZD inhibition of aromatase plays a role in their effects on bone metabolism. Mouse osteoblasts were cultured with and without HGC, and incubated in a medium with or without testosterone, pioglitazone or rosiglitazone. Cell growth, oleic acid uptake, alkaline phosphatase activity, and osteocalcin production were measured. TZDs inhibited estradiol production by up to 84% in HGC/mouse osteoblast co-cultures. TZDs induced mouse osteoblast death and increased oleic acid uptake. TZDs also inhibited alkaline phosphatase activity (58-75%, p<0.046) and osteocalcin production (52-75%, p<0.031). For all the parameters, there were no significant differences between the osteoblast cultures alone and the HCG/osteoblast co-cultures. TZD effects on osteoblast viability, oleic acid uptake, alkaline phosphatase and osteocalcin production are independent of their effects on aromatase.

Overexpression of Uromodulin-like1 accelerates follicle depletion and subsequent ovarian degeneration.

Murine Uromodulin-like 1 (Umodl1) encodes Ca(2+)-dependent EGF-like membrane-bound proteins. This study presents its novel expression in the immune and female reproductive systems. Upon stimulation by CD3/CD28 antibodies, Umodl1 showed a prompt and robust response in the proliferating CD4(+) T cells, suggesting its implication in immune defense against pathogens. In ovary, Umodl1 is regulated by gonadotropins. Mice carrying extra copies of functional Umodl1 were generated by BAC transgenesis. Defects in the female reproductive system became evident from 4 months of age, manifested by reduced or diminished fertility. Histology revealed that the ovaries contained very few discernible follicles in the cortical region, and were devoid of distinguishable corpus lutea (CL). Among the multilayered preantral follicles, elevated apoptosis was observed in both the oocytes and surrounding granulosa cells (GCs). Furthermore, a high level of PPARγ indicated an abnormal adipogenesis in the mutant ovaries, which resulted in the conversion of GCs into adipocytes. By 6 months of age, all mutant mice became anovulatory. Ovarian tissues including CL, follicles of various stages and associated stromal cells were degenerated. Altered expression of AMH, follicle-stimulating hormone and other ovary-specific marker genes such as Gdf-9, Rnf35, NOHLH and Gcx-1 further demonstrated that the molecular properties of the mutant ovaries have been severely disturbed. This work presents a novel animal model for investigating the pathogenesis of premature ovarian failure or early ovarian ageing.

Rosiglitazone and pioglitazone inhibit estrogen synthesis in human granulosa cells by interfering with androgen binding to aromatase.

The effects of rosiglitazone or pioglitazone (thiazolidinediones, TZDs) on estrogen production and aromatase activity in human ovarian cells were examined. Human granulosa cells were incubated in the tissue culture medium supplemented with androstenedione or testosterone, with or without insulin, TZDs, or type 1 17ß-hydroxysteroid-dehydrogenase (17ß-HSD) inhibitor. Estrogen concentrations in the conditioned medium, aromatase mRNA and protein expression in the cells and androgen substrate binding to aromatase were measured. With androstenedione as substrate, rosiglitazone or pioglitazone inhibited estrone production by up to 22% (p<0.012) while type 1 17ß-HSD inhibitor enhanced this effect of rosiglitazone or pioglitazone by 37% (p<0.001) and by 67% (p<0.001), respectively. With testosterone as substrate, rosiglitazone or pioglitazone inhibited estradiol production by 32% (p<0.001). With (3)H-testosterone as substrate, rosiglitazone or pioglitazone inhibited the (3)H-tritiated water release by the cultured cells by 45% and 35%, respectively, thus directly demonstrating inhibition of aromatase. Rosiglitazone or pioglitazone, however, had no significant effect on aromatase mRNA or protein expression. Rosiglitazone or pioglitazone inhibited (125)I-androstenedione and (125)I-testosterone binding to aromatase by 38% (p<0.001). It was concluded that rosiglitazone or pioglitazone inhibit estrogen synthesis in human granulosa cells by interfering with androgen binding to aromatase.

Vitamin D regulates steroidogenesis and insulin-like growth factor binding protein-1 (IGFBP-1) production in human ovarian cells.

Vitamin D Receptor (VDR) is expressed in both animal and human ovarian tissue, however, the role of vitamin D in human ovarian steroidogenesis is unknown. Cultured human ovarian cells were incubated in tissue culture medium supplemented with appropriate substrates, with or without 50 pM-150 pM or 50 nM-150 nM of 1,25-(OH)2D3, and in the presence or absence of insulin. Progesterone, testosterone, estrone, estradiol, and IGFBP-1 concentrations in conditioned tissue culture medium were measured. Vitamin D receptor was present in human ovarian cells. 1,25-(OH)2D3 stimulated progesterone production by 13% (p<0.001), estradiol production by 9% (p<0.02), and estrone production by 21% (p<0.002). Insulin and 1,25-(OH)2D3 acted synergistically to increase estradiol production by 60% (p<0.005). 1,25-(OH)2D3 alone stimulated IGFBP-1 production by 24% (p<0.001), however, in the presence of insulin, 1,25-(OH)2D3 enhanced insulin-induced inhibition of IGFBP-1 production by 13% (p<0.009). Vitamin D stimulates ovarian steroidogenesis and IGFBP-1 production in human ovarian cells likely acting via vitamin D receptor. Insulin and vitamin D synergistically stimulate estradiol production. Vitamin D also enhances inhibitory effect of insulin on IGFBP-1 production.

Treatment options for impaired spermatogenesis: germ cell transplantation and stem-cell based therapy.

Advances in infertility treatment had the most extraordinary breakthrough with the birth of the first in vitro fertilization baby in 1978. Fourteen years later, intracytoplasmic sperm injection has been introduced for the treatment of male factor infertility. Intra cytoplasmic sperm injection in combination with testicular sperm extraction has allowed men with azoospermia to father children. In fact, as long as a fully developed spermatozoon is identified, it can be utilized or can even be duplicated to inseminate several oocytes while providing information on its genomic content. There are, however, men who are suffering from spermatogenic arrest, where no post-meiotic germ cells are retrieved, and therefore, unable to generate their own offspring. More recently, the successful isolation and cultivation of spermatogonial stem cells has allowed the exploration of their biological characteristics and their application in therapeutic approaches following transplantation or in vitro maturation. Finally, men diagnosed with germ cell aplasia can only be treated by donor or de novo generated gametes. In the past several years, we have attempted to manufacture gametes by inducing haploidization of somatic cells and more recently, generating sperm-like cells through embryonic stem cell differentiation.

Histologic characteristics of the endometrium predicts success when utilizing autologous endometrial coculture in patients with IVF failure.

PURPOSE: To analyze the success of autologous endometrial coculture (AECC) in improving embryo quality and pregnancy outcome based on the histologic characteristic of the biopsy. METHODS: Prospective study of 86 consecutive patients undergoing IVF utilizing AECC. RESULTS: The patients were on average 37.4+/-4.0 years with a history of 2.6+/-1.8 failed previous attempts. An overall clinical pregnancy rate of 45.3% per ET was found. The embryos grown in AECC were of an improved quality in comparison to those grown in conventional media. 33.7% (29/86) of the biopsies were out of phase (>3 days). In-phase (IP) and OOP (out of phase) specimens both demonstrated an improvement in embryo quality. However, OOP endometrial biopsies that displayed significant retarded endometrial development (< cycle day 19) did not demonstrate an improvement in embryos grown on AECC as compared to IP endometrial biopsies or OOP endometrial biopsies that demonstrated at least an endometrial development of cycle day 19. CONCLUSIONS: We have demonstrated a significant improvement in embryo quality with AECC. We have also demonstrated that histologic dating of the endometrium is predictive of IVF outcome when utilizing AECC.

Intracytoplasmic sperm injection. Accomplishments and qualms.

AIM: Intracytoplasmic sperm injection (ICSI) is now the preferred technique for treatment of male factor infertility and many children have been born worldwide. However, concerns about the risk of transmitting genetic defects and development of ICSI children have been raised. We report clinical outcome of ICSI in Cornell University and results of screening for genetic defects in ICSI parents and their children. METHODS: Pregnancy and obstetrical outcomes as well as congenital malformations were analyzed. Chromosomal karyotyping and Yq deletion assessments were performed on blood samples. In addition, medical and developmental outcome were assessed in 3 and 5 year old ICSI children. RESULTS: We have performed 8 575 ICSI cycles with ejaculated spermatozoa that resulted in a 75.4% fertilization and a 42.8% clinical pregnancy rates while for surgically retrieved specimen resulted in 66%, 48.8% respectively. The incidence of Y deletion was within the expected range for an infertile population, with identical deletions transmitted to the male offspring. There were no differences in cognitive, motor and behavioral development observed between ICSI children and these conceived naturally. CONCLUSION: The large majority of infertile men were treatable by ICSI, which resulted in the transmission of a specific abnormality but did not enhance the incidence of de novo deletions. There is no indication that ICSI children develop more congenital defects or express a lower psychomotor development that children conceived naturally. Nonetheless, genetic screening and counseling of couples undergoing ICSI would seem to be appropriate.

Maternal serum levels of interferon-gamma and interleukin-2 soluble receptor-alpha predict the outcome of early IVF pregnancies.

BACKGROUND: Elevated maternal serum levels of interleukin-2 soluble receptor-alpha (IL-2 sRalpha), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) have been associated with pregnancy loss. The aim of our study was to evaluate the predictive value of these cytokines in the outcome of early IVF pregnancies. METHODS: One hundred and fifty-nine consecutive IVF patients who were subsequently diagnosed to have a biochemical pregnancy (n = 23), a first-trimester miscarriage (n = 19) or a normal term delivery (n = 117) were included in this study. Serum was collected from the initial pregnancy test, 11 days after a day 3 embryo transfer, and all samples were analysed for IL-2 sRalpha, TNF-alpha and IFN-gamma by commercially available enzyme-linked immunosorbent assay (ELISA) kits. RESULTS: IL-2 sRalpha levels were significantly higher in patients with an early pregnancy loss compared with patients with a normal term delivery (849.5 +/- 69.6 versus 693.5 +/- 31.2 pg/ml, P = 0.02), and a cut-off point of IL-2 sRalpha >1000 pg/ml predicted a poor pregnancy outcome (44.4 versus 22.7% pregnancy loss, IL-2 sRalpha >or=1000 versus IL-2 sRalpha <1000 pg/ml; P = 0.02). IFN-gamma-positive patients had twice the risk for poor IVF pregnancy outcome compared with IFN-gamma-negative subjects (40.8 versus 20.0%, respectively; P < 0.02), including a significantly lower implantation rate (37.6 +/- 0.05 versus 50.0 +/- 0.03%, respectively; P = 0.02). There was no difference in pregnancy outcome based upon serum levels, or the ability to detect the presence of TNF-alpha. No differences in levels of these cytokines were found based on the aetiology of the patients' infertility. CONCLUSIONS: Elevated maternal serum levels of IL-2 sRalpha and IFN-gamma as early as 11 days after embryo transfer are associated with poor IVF pregnancy outcome.

Application of intracytoplasmic sperm injection in assisted reproductive technologies.

Intracytoplasmic sperm injection (ICSI) entails the mechanical insertion of a chosen spermatozoon directly into the cytoplasm of an oocyte. Due to the consistent fertilization and pregnancy outcome, ICSI is routinely used to treat azoospermic patients where spermatozoa are retrieved by epididymal aspiration or testicular biopsy. Since male subfertility has been associated with a higher incidence of genomic defects, ranging from numerical chromosomal abnormalities to Yq microdeletions, concerns have been raised as to the risk of transmitting genetic defects to the offspring. Screening for such defects can provide invaluable information for appropriate counselling prior to ICSI treatment. In order to address these concerns, a follow-up of the children born after ICSI treatment was conducted.

Detection of sperm in men with Y chromosome microdeletions of the AZFa, AZFb and AZFc regions.

BACKGROUND: Y chromosome microdeletions are associated with severe male factor infertility. In this study, the success rate of testicular sperm retrieval was determined for men with deletions of AZF regions a, b or c. METHODS: AZF deletions were detected by PCR of 30 sequence-tagged sites within Yq emphasizing the AZFa, b and c regions. Semen analysis and diagnostic testis biopsy or testicular sperm extraction (TESE) findings were correlated with the specific AZF region deleted. RESULTS: A total of 78 men with AZF deletions included three with AZFa deletion, 11 with AZFb, 42 with AZFc, 16 with AZFb+c and six with Yq (AZFa+b+c). All men with AZFa, AZFb, AZFb+c and Yq deletions were azoospermic and no sperm were found with TESE or biopsy. Of men with isolated AZFc deletion, sperm were found in 75% (9/12) by TESE and 45% (9/20) on biopsy (56% overall); 62% (26/42) were azoospermic and 38% (16/42) severely oligozoospermic. A total of 7 patients with deletion patterns that included the complete AZFa region and 23 that included the complete AZFb region who underwent TESE or biopsy did not have sperm detected by these surgical measures. CONCLUSIONS: Microdeletion of the entire AZFa or AZFb regions of the Y chromosome portends an exceptionally poor prognosis for sperm retrieval, whereas the majority of men with AZFc deletion have sperm within the semen or testes available for use in IVF/ICSI.