Classification of Atretic Small Antral Follicles in the Human Ovary.

The reproductive lifespan in humans is regulated by a delicate cyclical balance between follicular recruitment and atresia in the ovary. The majority of the small antral follicles present in the ovary are progressively lost through atresia without reaching dominance, but this process remains largely underexplored. In our study, we investigated the characteristics of atretic small antral follicles and proposed a classification system based on molecular changes observed in granulosa cells, theca cells, and extracellular matrix deposition. Our findings revealed that atresia spreads in the follicle with wave-like dynamics, initiating away from the cumulus granulosa cells. We also observed an enrichment of CD68+ macrophages in the antrum during the progression of follicular atresia. This work not only provides criteria for classifying three stages of follicular atresia in small antral follicles in the human ovary but also serves as a foundation for understanding follicular degeneration and ultimately preventing or treating premature ovarian failure. Understanding follicular remodeling in the ovary could provide a means to increase the number of usable follicles and delay the depletion of the follicular reserve, increasing the reproductive lifespan.

Dynamic in vitro culture of cryopreserved-thawed human ovarian cortical tissue using a microfluidics platform does not improve early folliculogenesis.

Current strategies for fertility preservation include the cryopreservation of embryos, mature oocytes or ovarian cortical tissue for autologous transplantation. However, not all patients that could benefit from fertility preservation can use the currently available technology. In this regard, obtaining functional mature oocytes from ovarian cortical tissue in vitro would represent a major breakthrough in fertility preservation as well as in human medically assisted reproduction. In this study, we have used a microfluidics platform to culture cryopreserved-thawed human cortical tissue for a period of 8 days and evaluated the effect of two different flow rates in follicular activation and growth. The results showed that this dynamic system supported follicular development up to the secondary stage within 8 days, albeit with low efficiency. Surprisingly, the stromal cells in the ovarian cortical tissue were highly sensitive to flow and showed high levels of apoptosis when cultured under high flow rate. Moreover, after 8 days in culture, the stromal compartment showed increase levels of collagen deposition, in particular in static culture. Although microfluidics dynamic platforms have great potential to simulate tissue-level physiology, this system still needs optimization to meet the requirements for an efficient in vitro early follicular growth.

Previous caesarean section is associated with lower subsequent in vitro fertilization live birth rates.

This retrospective cohort study examines the association between previous mode of delivery and subsequent live birth rate in women who become pregnant after in vitro fertilization (IVF) or intra cytoplasmic sperm injection (ICSI) after their first delivery. The study included 112 women with a previous caesarean section and 418 women with a previous vaginal delivery, and a total of 1588 embryo transfers between January 2005 and June 2016 (Leiden University Medical Centre, the Netherlands). The mean age was 35 years and mean number of embryos transferred per attempt, 1.18. The study population included a total of 429 pregnancies resulting in 296 live births. The crude odds ratio for a subsequent live birth per embryo transfer was 0.60 (CI; 0.44 to 0.83, p = 0.002) in women with a previous caesarean section compared to women with a previous vaginal delivery. After adjustment for age, fresh/frozen-thawed embryo transfer and quality of the embryo, the odds ratio was 0.64 (CI; 0.46 to 0.89, p = 0.01). It was concluded that in subfertile women trying to achieve a subsequent pregnancy with IVF or ICSI, a history of caesarean section was associated with a reduced live birth rate per embryo transfer compared to women with a history of one previous vaginal delivery.

Single-Cell Transcriptomics Analysis of Human Small Antral Follicles.

Human ovarian folliculogenesis is a highly regulated and complex process. Characterization of follicular cell signatures during this dynamic process is important to understand follicle fate (to grow, become dominant, or undergo atresia). The transcriptional signature of human oocytes and granulosa cells (GCs) in early-growing and ovulatory follicles have been previously described; however, that of oocytes with surrounding GCs in small antral follicles have not been studied yet. Here, we have generated a unique dataset of single-cell transcriptomics (SmartSeq2) consisting of the oocyte with surrounding GCs from several individual (non-dominant) small antral follicles isolated from adult human ovaries. We have identified two main types of (healthy) follicles, with a distinct oocyte and GC signature. Using the CellphoneDB algorithm, we then investigated the bi-directional ligand-receptor interactions regarding the transforming growth factor-ß (TGFß)/bone morphogenetic protein (BMP), wingless-type (MMTV)-integration site (WNT), NOTCH, and receptor tyrosine kinases (RTK) signaling pathways between oocyte and GCs within each antral follicle type. Our work not only revealed the diversity of small antral follicles, but also contributes to fill the gap in mapping the molecular landscape of human folliculogenesis and oogenesis.

Long-Term Risk of Ovarian Cancer and Borderline Tumors After Assisted Reproductive Technology.

BACKGROUND: Long-term effects of assisted reproductive technology (ART) on ovarian tumor risk are unknown. METHODS: This nationwide cohort study comprises 30 625 women who received ovarian stimulation for ART in 1983-2000 and 9988 subfertile women not treated with ART. Incident invasive and borderline ovarian tumors were ascertained through linkage with the Netherlands Cancer Registry and the Dutch Pathology Registry until July 2018. Ovarian tumor risk in ART-treated women was compared with risks in the general population and the subfertile non-ART group. Statistical tests were 2-sided. RESULTS: After a median follow-up of 24 years, 158 invasive and 100 borderline ovarian tumors were observed. Ovarian cancer risk in the ART group was increased compared with the general population (standardized incidence ratio [SIR] = 1.43, 95% confidence interval [CI] = 1.18 to 1.71) but not when compared with the non-ART group (age- and parity-adjusted hazard ratio [HR] = 1.02, 95% CI = 0.70 to 1.50). Risk decreased with higher parity and with a larger number of successful ART cycles (resulting in childbirth, Ptrend = .001) but was not associated with the number of unsuccessful ART cycles. Borderline ovarian tumor risk was increased in ART-treated women compared with the general population (SIR = 2.20, 95% CI = 1.66 to 2.86) and with non-ART women (HR = 1.84, 95% CI = 1.08 to 3.14). Risk did not increase with more ART cycles or longer follow-up time. CONCLUSIONS: Increased ovarian cancer risk in ART-treated women compared with the general population is likely explained by nulliparity rather than ART treatment. The increased risk of borderline ovarian tumors after ART must be interpreted with caution because no dose-response relationship was observed.

Ovarian tissue cryopreservation: Low usage rates and high live-birth rate after transplantation.

INTRODUCTION: The likelihood of survival after cancer treatment among young women with cancer has increased considerably, quality of life after treatment has drawn more attention. However, in young fertile women, fertility preservation is an important issue with regard to quality of life. One of the options of fertility preservation is ovarian tissue cryopreservation. The purpose of this follow-up study is to present our clinical experiences and evaluate the long-term follow up of ovarian cryopreservation to improve future patient selection. MATERIAL AND METHODS: From July 2002 to December 2015 at the Leiden University Hospital, the Netherlands, 69 young women underwent ovarian tissue cryopreservation when they were at risk of iatrogenic premature ovarian insufficiency. Follow-up data with regard to ovarian function were obtained until October 2018, from medical records and questionnaires. RESULTS: Of the 69 women in whom ovarian tissue cryopreservation was performed, 12 died (15.9%), 57 were approached to participate, of which 6 were lost to follow up. The indications for ovarian tissue cryopreservation were malignant (81.1%) and benign (18.9%) diseases in which gonadotoxic treatment was scheduled. In total, twenty women (39.2%) are known to have premature ovarian insufficiency due to gonadotoxic treatment. Fifteen women conceived spontaneously, and delivered 25 babies. In this cohort, the usage rate of autotransplantation is 8.7% (7/69). In total, nine autotransplantations of cryopreserved ovarian tissue were performed in seven patients (of which 1 ovarian tissue cryopreservation was performed in another hospital) after which 6 babies were born to four women, giving a live-birth rate of 57%. CONCLUSIONS: Ovarian tissue cryopreservation followed by autotransplantation is an effective method to restore fertility (live-birth rate of 57%). The usage rate of 8.7% (6/69) indicates that more knowledge about the risk of premature ovarian insufficiency after gonadotoxic treatment is needed to be able to offer ovarian tissue cryopreservation more selectively.

At Term, XmO and XpO Mouse Placentas Show Differences in Glucose Metabolism in the Trophectoderm-Derived Outer Zone.

Genetic mouse model (39,XO) for human Turner Syndrome (45,XO) harboring either a single maternally inherited (Xm) or paternally inherited (Xp) chromosome show a pronounced difference in survival rate at term. However, a detailed comparison of XmO and XpO placentas to explain this difference is lacking. We aimed to investigate the morphological and molecular differences between XmO and XpO term mouse placentas. We observed that XpO placentas at term contained a significantly larger area of glycogen cells (GCs) in their outer zone, compared to XmO, XX, and XY placentas. In addition, the outer zone of XpO placentas showed higher expression levels of lactate dehydrogenase (Ldha) than XmO, XX, and XY placentas, suggestive of increased anaerobic glycolysis. In the labyrinth, we detected significantly lower expression level of trophectoderm (TE)-marker keratin 19 (Krt19) in XpO placentas than in XX placentas. The expression of other TE-markers was comparable as well as the area of TE-derived cells between XO and wild-type labyrinths. XpO placentas exhibited specific defects in the amount of GCs and glucose metabolism in the outer zone, suggestive of increased anaerobic glycolysis, as a consequence of having inherited a single Xp chromosome. In conclusion, the XpO genotype results in a more severe placental phenotype at term, with distinct abnormalities regarding glucose metabolism in the outer zone.

Human Extravillous Trophoblasts Penetrate Decidual Veins and Lymphatics before Remodeling Spiral Arteries during Early Pregnancy.

In humans, the defective invasion of the maternal endometrium by fetal extravillous trophoblasts (EVTs) can lead to insufficient perfusion of the placenta, resulting in pregnancy complications that can put both mother and baby at risk. To study the invasion of maternal endometrium between (W)5.5-12 weeks of gestation by EVTs, we combined fluorescence in situ hybridization, immunofluorescence and immunohistochemistry to determine the presence of (male) EVTs in the vasculature of the maternal decidua. We observed that interstitial mononuclear EVTs directly entered decidual veins and lymphatics from W5.5. This invasion of decidual veins and lymphatics occurred long before endovascular EVTs remodelled decidual spiral arteries. This unexpected early entrance of interstitial mononuclear EVTs in the maternal circulation does not seem to contribute to the materno-placental vascular connection directly, but rather to establish (and expand) the materno-fetal interface through an alternative vascular route.

Ovarian Stimulation for In Vitro Fertilization and Long-term Risk of Breast Cancer.

IMPORTANCE: Previous studies of breast cancer risk after in vitro fertilization (IVF) treatment were inconclusive due to limited follow-up. OBJECTIVE: To assess long-term risk of breast cancer after ovarian stimulation for IVF. DESIGN, SETTING, AND PARTICIPANTS: Historical cohort (OMEGA study) with complete follow-up through December 2013 for 96% of the cohort. The cohort included 19,158 women who started IVF treatment between 1983 and 1995 (IVF group) and 5950 women starting other fertility treatments between 1980 and 1995 (non-IVF group) from all 12 IVF clinics in the Netherlands. The median age at end of follow-up was 53.8 years for the IVF group and 55.3 years for the non-IVF group. EXPOSURES: Information on ovarian stimulation for IVF, other fertility treatments, and potential confounders was collected from medical records and through mailed questionnaires. MAIN OUTCOMES AND MEASURES: Incidence of invasive and in situ breast cancers in women who underwent fertility treatments was obtained through linkage with the Netherlands Cancer Registry (1989-2013). Breast cancer risk in the IVF group was compared with risks in the general population (standardized incidence ratios [SIRs]) and the non-IVF group (hazard ratios [HRs]). RESULTS: Among 25,108 women (mean age at baseline, 32.8 years; mean number of IVF cycles, 3.6), 839 cases of invasive breast cancer and 109 cases of in situ breast cancer occurred after a median follow-up of 21.1 years. Breast cancer risk in IVF-treated women was not significantly different from that in the general population (SIR, 1.01 [95% CI, 0.93-1.09]) and from the risk in the non-IVF group (HR, 1.01 [95% CI, 0.86-1.19]). The cumulative incidences of breast cancer at age 55 were 3.0% for the IVF group and 2.9% for the non-IVF group (P = .85). The SIR did not increase with longer time since treatment (≥20 years) in the IVF group (0.92 [95% CI, 0.73-1.15]) or in the non-IVF group (1.03 [95% CI, 0.82-1.29]). Risk was significantly lower for those who underwent 7 or more IVF cycles (HR, 0.55 [95% CI, 0.39-0.77]) vs 1 to 2 IVF cycles and after poor response to the first IVF cycle (HR, 0.77 [95% CI, 0.61-0.96] for <4 vs ≥4 collected oocytes). CONCLUSIONS AND RELEVANCE: Among women undergoing fertility treatment in the Netherlands between 1980 and 1995, IVF treatment compared with non-IVF treatment was not associated with increased risk of breast cancer after a median follow-up of 21 years. Breast cancer risk among IVF-treated women was also not significantly different from that in the general population. These findings are consistent with absence of a significant increase in long-term risk of breast cancer among IVF-treated women.

On the development of extragonadal and gonadal human germ cells.

Human germ cells originate in an extragonadal location and have to migrate to colonize the gonadal primordia at around seven weeks of gestation (W7, or five weeks post conception). Many germ cells are lost along the way and should enter apoptosis, but some escape and can give rise to extragonadal germ cell tumors. Due to the common somatic origin of gonads and adrenal cortex, we investigated whether ectopic germ cells were present in the human adrenals. Germ cells expressing DDX4 and/or POU5F1 were present in male and female human adrenals in the first and second trimester. However, in contrast to what has been described in mice, where 'adrenal' and 'ovarian' germ cells seem to enter meiosis in synchrony, we were unable to observe meiotic entry in human 'adrenal' germ cells until W22. By contrast, 'ovarian' germ cells at W22 showed a pronounced asynchronous meiotic entry. Interestingly, we observed that immature POU5F1+ germ cells in both first and second trimester ovaries still expressed the neural crest marker TUBB3, reminiscent of their migratory phase. Our findings highlight species-specific differences in early gametogenesis between mice and humans. We report the presence of a population of ectopic germ cells in the human adrenals during development.

Stronger T-Cell Alloreactivity and Diminished Suppressive Capacity of Peripheral Regulatory T Cells in Infertile Women Undergoing In Vitro Fertilization.

PROBLEM: Increasing evidence suggests modulation of the maternal immune response to be essential for successful pregnancy. We studied the immunophenotypic profile and function of peripheral blood T lymphocytes in infertile women undergoing in vitro fertilization (IVF) and fertile control population. METHOD: We collected peripheral blood mononuclear cells (PBMC) from infertile patients with recurrent implantation failure (RIF), infertile patients with successful IVF (IVFs), and normal fertile women. Cells were phenotypically analyzed, and the proliferative response and cytokine production were studied in mixed lymphocyte cultures (MLC), using lymphocytes of the own partner, or a third-party male as stimulators cells. To examine the suppressive capacity of regulatory regulatory T cells (Tregs), we performed MLC studies with a CD45(+) fraction depleted for CD4(+) CD25(bright) T cells. RESULTS: No significant differences in proportions of subsets of circulating T lymphocytes were observed. The proliferative allo-immune response of PBMC of IVF women (RIF and IVFs) was significantly higher, with higher production of T-helper cells (Th1) and Th2 cytokines, compared to the fertile women. This difference in proliferation and cytokine production was associated with a diminished suppressive capacity of Tregs in these women. CONCLUSION: The higher allo-immune response of the IVF women compared to fertile women might be the result of a diminished suppressive capacity of Tregs and emphasizes the important role of Tregs during conception.

Piwi proteins and piRNAs in mammalian oocytes and early embryos.

Germ cells of most animals critically depend on piRNAs and Piwi proteins. Surprisingly, piRNAs in mouse oocytes are relatively rare and dispensable. We present compelling evidence for strong Piwi and piRNA expression in oocytes of other mammals. Human fetal oocytes express PIWIL2 and transposon-enriched piRNAs. Oocytes in adult human ovary express PIWIL1 and PIWIL2, whereas those in bovine ovary only express PIWIL1. In human, macaque, and bovine ovaries, we find piRNAs that resemble testis-borne pachytene piRNAs. Isolated bovine follicular oocytes were shown to contain abundant, relatively short piRNAs that preferentially target transposable elements. Using label-free quantitative proteome analysis, we show that these maturing oocytes strongly and specifically express the PIWIL3 protein, alongside other, known piRNA-pathway components. A piRNA pool is still present in early bovine embryos, revealing a potential impact of piRNAs on mammalian embryogenesis. Our results reveal that there are highly dynamic piRNA pathways in mammalian oocytes and early embryos.

Development of the follicular basement membrane during human gametogenesis and early folliculogenesis.

BACKGROUND: In society, there is a clear need to improve the success rate of techniques to restore fertility. Therefore a deeper knowledge of the dynamics of the complex molecular environment that regulates human gametogenesis and (early) folliculogenesis in vivo is necessary. Here, we have studied these processes focusing on the formation of the follicular basement membrane (BM) in vivo. RESULTS: The distribution of the main components of the extracellular matrix (ECM) collagen IV, laminin and fibronectin by week 10 of gestation (W10) in the ovarian cortex revealed the existence of ovarian cords and of a distinct mesenchymal compartment, resembling the organization in the male gonads. By W17, the first primordial follicles were assembled individually in that (cortical) mesenchymal compartment and were already encapsulated by a BM of collagen IV and laminin, but not fibronectin. In adults, in the primary and secondary follicles, collagen IV, laminin and to a lesser extent fibronectin were prominent in the follicular BM. CONCLUSIONS: The ECM-molecular niche compartimentalizes the female gonads from the time of germ cell colonization until adulthood. This knowledge may contribute to improve methods to recreate the environment needed for successful folliculogenesis in vitro and that would benefit a large number of infertility patients.

Perinatal outcome, health, growth, and medical care utilization of 5- to 8-year-old intracytoplasmic sperm injection singletons.

OBJECTIVE: To evaluate short- and long-term health in intracytoplasmic sperm injection (ICSI) singletons. DESIGN: Follow-up study. SETTING: University medical center, assessments between March 2004 and May 2005. PATIENT(S): Singletons born between June 1996 and December 1999 after ICSI in the Leiden University Medical Center laboratory were compared with matched singletons born after IVF and natural conception. INTERVENTION(S): Mode of conception. MAIN OUTCOME MEASURE(S): An examiner blinded to the conception mode of the child assessed congenital malformations and growth. Information on pregnancy, perinatal period, birth defects, general health, and medical consumption was obtained through questionnaires. RESULT(S): Outcomes of children conceived by ICSI and IVF (n = 81/81, preterm infants excluded) were comparable or even more positive for ICSI. Perinatal outcomes were poorer after ICSI than natural conception: prematurity: P=.014; low birth weight: odds ratio = 7.4, 95% confidence interval (CI) [0.9; 62.5]; mean birth weight: Delta = 186 g, 95% CI [21; 351]. The ICSI mothers had more pregnancy complications (n = 33 vs. 18) and in-hospital deliveries (prevalence ratio 1.36, 95% CI 1.17; 1.48). No further differences were found between ICSI and natural conception children on congenital malformations, health, growth, and medical consumption (n = 87/85, preterm infants included). CONCLUSION(S): No adverse health outcomes were identified in ICSI singletons up to age 5-8 years compared to IVF and natural conception singletons, besides poorer perinatal outcomes after ICSI versus natural conception.

Cognitive development of singletons born after intracytoplasmic sperm injection compared with in vitro fertilization and natural conception.

OBJECTIVE: To investigate cognitive development of singletons conceived by intracytoplasmic sperm injection (ICSI) at 5-8 years of age. DESIGN: Follow-up study. SETTING: University medical center, assessments between March 2004 and May 2005. PATIENT(S): Singletons born between June 1996 and December 1999 after ICSI at the Leiden University Medical Center were compared with matched singletons born after IVF and natural conception (NC). INTERVENTION(S): Mode of conception. MAIN OUTCOME MEASURE(S): Intelligence quotient (IQ) was measured with the Revised Amsterdam Child Intelligence Test (short form). The investigators were blinded to conception mode. RESULT(S): Singletons conceived by ICSI (n = 83) achieved lower IQ scores than IVF singletons (n = 83) (adjusted mean difference IQ: 3.6 [95% confidence interval (CI) -0.8, 8.0]). After categorizing IQ outcomes (<85, 85-115, >115), no significant difference in the distribution of IQ was found. Singletons conceived by ICSI (n = 86) achieved lower IQ scores than NC singletons (n = 85); the adjusted mean difference varied between 5 and 7 points (5.6 [95% CI 0.9, 10.3]; 7.1 [95% CI 1.7, 12.5]) depending on the covariates included in the model. Adjustment for prematurity did not change the results. Percentages in IQ categories <85, 85-115, and >115 were 12%, 64%, and 24% for ICSI and 6%, 54%, and 40% for NC, respectively. CONCLUSION(S): In the relatively limited sample investigated, cognitive development among ICSI singletons was lower than among IVF and NC singletons. Infertility factors or unmeasured confounders may play a role.

Matched follow-up study of 5-8 year old ICSI-singletons: comparison of their neuromotor development to IVF and naturally conceived singletons.

BACKGROUND: Intracytoplasmic sperm injection (ICSI) is an invasive technique of artificial reproduction. We investigated the effect of ICSI on neuromotor development in 5-8 year old singletons. METHODS: We did a follow-up of ICSI-singletons born between 1996 and 1999 after treatment in the Leiden University Medical Center and compared them with matched controls born after in vitro fertilization (IVF) and natural conception (NC). Children underwent a thorough neurological examination that focused on minor neurological dysfunction (MND). RESULTS: There were no differences in outcome between ICSI (n = 81) and IVF-children (n = 81), all born at term: MND prevalence 66.3% versus 61.3%, prevalence ratio (PR) 1.08 [0.83; 1.29]. MND prevalence among all ICSI-children (n = 87) was higher than among NC-controls (n = 85) (66.3% versus 50.6%, PR 1.31 [1.02; 1.55]). After adjustment for maternal age and parity, the PR remained elevated but was no longer statistically significant (adjusted PR 1.22 [0.86; 1.52]). When comparing only term ICSI and NC-children (n = 81; n = 85), the PR adjusted for maternal age and parity was 1.20 [0.83; 1.51]. CONCLUSIONS: Neuromotor outcome of 5-8 year old singletons born at term after ICSI or IVF was similar; ICSI-children (both the total group and term children only) deviated slightly from NC-controls. Part of this effect was explained by a difference in parity, but not prematurity.