Authors: Paula Brígido, Shuyana Deba, Javier Del Río and Isabel Sánchez
Implantation is one of the key steps in human reproduction, and hatching of the blastocyst is a critical point in the sequence of physiological events that lead to implantation (2). It has been estimated that only 15-20% of embryo transfers culminate in implantation, and as a consequence clinical pregnancy and live birth rates are quite low (3, 4). The potential of an embryo to implant is related to its own nature, the quality of gametes and the endometrial receptivity. But failure in blastocyst hatching is also an important factor that prevents implantation (2, 3, 4). With the aim of solving this problem and in order to improve both implantation and pregnancy rates after Assisted Reproductive Technology (ART) procedures, scientists developed assisted hatching (AH) (2, 3, 4). AH consists of an artificial alteration of the zona pellucida (ZP) either by slimming or breakage. This technique was first described on 1988 by Cohen et al., who reported the first pregnancy after AH (4), and successive works have since shown its efficiency (2, 3).
Because AH does not seem to present clear advantages to all patients, it should not be applied, in principle, to all of them as a rule (3). Some studies have shown that AH has effectively improved ART outcomes in patients with bad prognosis, like those with a history of 2+ implantation failures, bad embryo quality, aged patients (38+), patient with frozen embryos or those who have oocytes with a thick ZP (2, 3, 4, 6).
ART have some actual consequences on gametes and embryos, one of them being hardening of the ZP by the use of certain culture media or by cryopreservation. These and other negative effects may hamper blastocyst hatching, which might be solved through AH. Overall, this approach might be useful considering the early implantation window in women treated with exogenous gonadotrophin stimulation, compared to a natural cycle. Additionally, benefits from AH include the possibility of ZP breakage improving the embryo-endometrium communication (5).
In order to help the embryo hatch through the ZP before implantation, a variety of techniques over the years have been developed (7). As a commonality for all of them, it is important to minimize the time of the embryo being outside the incubator and to optimize the methodologies to reduce both pH and temperature variations (3).
MECHANICAL ASSISTED HATCHING (PARTIAL ZONA DISSECTION)
This method does not require any new equipment, and it can be easily performed by an embryologist handling a micromanipulator (8). During this procedure, the embryo is held by the suction exerted by the holding pipette, while the microneedle is passed through the zona pellucida at the largest visible portion of the perivitelline space and introduced in a tangential trajectory to the embryo. Then, in order to tear the ZP apart, the embryo is released from the holding pipette and rubbed against it (7).
However, one of the main disadvantages of this technique is the difficulty of creating a hole of significant size without abruptly damaging the embryo. Moreover, it is not a fast procedure, which means embryos need to remain outside the incubator for a long time, and so this may affect development of the transferred embryos (7, 9).
ACID TYRODE'S ASSISTED HATCHING
For this procedure, first the embryo is stabilized with the holding pipette. Second, a pipette containing acid Tyrode is approached to the embryo, oriented at the 3 o`clock position, next to an area of empty perivitelline space. Finally, the acidic solution is gently expelled over a small area (~30 µm Ø) and immediately washed away (7). This technique allows the formation of a hole of significant size; nevertheless, the exposure to acid is problematic, since this compound may be potentially embryotoxic. Furthermore, this technique requires practice and expertise from the operator (7, 10).
Laser-assisted hatching (LAH) is designed for easy positioning of the embryos, focus and shooting. It can be executed with a single click of the mouse controller. The diameter of the drilled holes vary between 5 and 10 µm according to how many shots are performed and the irradiation time (7). From the technical point of view, this method is easier, better controlled and more precise. Without physical contact with other embryos, the procedure can be completed faster than other methods. So, time of the embryos out of the incubator is shorter than for other techniques (9).
Even though the equipment may result expensive, the laser-assisted technique seems to represent the lowest potential risk for the embryo, and it is relatively simple to perform with consistency between operators (3).
EFFICACY OF AH
Although it has been performed for more than 20 years, up to date results are still inconclusive. Ma and co-authors performed one of the first trials to determine the overall effect of this technique. They concluded that implantation rates could be enhanced by performing ICSI along with AH, but differences were not statistically significant (11).
Two recent meta-analyses evaluating potential benefits of this technique have reported significant heterogeneity among results (12, 13), suggesting that effects of AH may differ depending on specific patient features (14).
Most researchers support the hypothesis that this technique improves clinical pregnancy rates in patients with previous failed IVF cycles or poor prognosis. However, there is insufficient evidence to affirm that AH improves live-birth rates in these populations, and so it remains uncertain whether AH is beneficial to other patients (6, 14, 16, 17).
IS THERE AN INCREASE IN MULTIPLE GESTATION RATE?
Even though certain reports associate artificial manipulation of the zona pellucida with multiple pregnancies, there is actually insufficient evidence to support an increased risk of monozygotic twinning after AH. In fact, the overall rate of monozygotic twin pregnancy in IVF with AH is less than 1% (6, 16).
MISCARRIAGE AND CONGENITAL MALFORMATIONS RATES
It has been reported that this technique may enhance implantation of abnormal embryos. Thus, the lower live birth rate observed, which is related to the high number of pregnancies ending in early miscarriage, may be due to chromosomal abnormalities of the embryos (17). However, Ma et al. performed cytogenetic tests on miscarriaged embryos and umbilical cord blood from newborn infants, where they found a similar incidence of major congenital malformations in ICSI-born patients compared to the general population (11), thus ruling out any direct relationship of AH and miscarriage.
EFFECTS OF ASSISTED HATCHING ON FROZEN CYCLES
Data show that cryopreservation may induce zona hardening as well as advanced female age and in vitro culture conditions (18). The answer to this problem could be AH, but results obtained on implantation and pregnancy rates after AH for frozen embryo transfer cycles are controversial, as well as for fresh IVF cycles (19). These discrepancies may be attributed to the type of AH, the extent of ZP microdissection, the number of patients and criteria for their selection, or even the quality and stage of embryos selected for AH performance.
In an earlier study, Primi et al. (2004) were unable to show any specific advantage of LAH in cryopreserved embryos. In this study, no embryo selection was reported, so this could explain why the implantation rates observed were lower than those achieved when embryos were selected (20).
Ng et al. (2005) also found a negative effect of LAH on frozen-thawed embryos in their randomised study (implantation rates: 9.0% vs. 12.5%; pregnancy rates: 6.8% vs. 15%). Although a subgroup analysis showed a higher implantation rate, differences were not statistically significant when LAH was performed on embryos with zona thickness of 1.6 mm (21).
In contrast, other studies using similar techniques of LAH were able to show improvement in implantation and pregnancy rates in the LAH group. Such was the case in a study by Balaban and co-authors (2006), in which their data revealed that implantation rates (20.1% vs. 9.9%) and pregnancy rates (40.9% vs. 27.3%) were significantly higher in the group in which embryos had been subjected to LAH before transfer, as compared to those from the control group (22). Valojerdi et al. (2008) in turn showed that LAH increased significantly both implantation and pregnancy rates in embryo cryopreservation cycles (23). These conclusions resemble those by Kanyo and co-authors (2016), who compared clinical pregnancy rates after using LAH technique on day-3 frozen-thawed embryos, and found a higher pregnancy rate after applying the LAH procedure (24).
Reports on patient populations are found that show benefits from AH, which include patients whose embryos present a thick zona, with elevated FSH, over 38 years old and cryopreserved cycles. It seems that the laser-based method is the most used among professionals and the most effective, although differences between are found between studies. However, no significant data show any increase in clinical pregnancy rates that could be translated into an encouragement of this technique to be routinely performed.
It should also be noted that some studies do not include specific important confounding factors, such as patient socioeconomic status, parity, duration of infertility, number of repeated failed cycles, embryo quality and/or smoking and alcohol intake, which might cause residual variance in the obtained results. Additional limitations of the study include the lack of information on the type of assisted hatching (mechanical, chemical, or laser), which may have changed over time or between clinics.
To learn more about this topic, visit our ‘VIDEOS’ section, where you will find videos about mechanical, chemical and laser hatching.
Authors: Shuyana Deba, Javier Del Río, Isabel Sánchez and Sara Sanz
Fertilization is a sequence of coordinated events that results in the metabolic activation of the ootid (nearly mature oocyte) and triggers cleavage of the zygote (2).
Nowadays, in assisted reproduction laboratories cleavage can be evaluated in vitro and in real time. Once in vitro fertilization (IVF) has been accomplished, early development of the embryo can be recorded by using time-lapse systems (TLP) (3). This approach makes it possible to evaluate morphology, including dynamic parameters, based on the uninterrupted culture of the embryo, which also allows for a better embryo selection, thus rising pregnancy rates (4). Even so, there are still clinics all over the world that select embryos for transfer using light microscopy, which means the use of a conventional incubation method (5).
CRITERIA FOLLOWED FOR EMBRYO CLASSIFICATION
It is known that an international consensus is needed in the way embryos are assessed and described. The following standardized criteria is from Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology, 2011 and includes ‘minimum standards’ for oocyte and embryo morphology scoring (6): the current expected observation for embryo development is 4 cells on day 2 and 8 cells on day 3 after fertilization (day 0). Moreover, embryos with <10% fragmentation, stage-specific cell size and not multinucleated are considered of good quality (6).
According to this consensus, scoring for day 4 (morula stage) regards as good embryos those that enter into a fourth round of cleavage, which implies evidences of compaction that virtually involve the whole volume of the embryo (6). Finally, on day 5 blastocysts are to be observed expanded with: a prominent inner cell mass (ICM) consisting of many cells, compacted and tightly adhered together; and a trophectoderm (TE), forming a cohesive epithelium (6).
Nevertheless, these parameters do not restrict laboratories to annotate further observations in order to select the best embryo for transfer (6).
BEST DAY TO PERFORM EMBRYO TRANSFER
One of the most important aspects that influence the success of ART is embryo transfer from the culture medium to the uterus (7). This has been a controversial subject that still generates quite some doubts. Morphological evaluation of embryos is sometimes a subjective process, and it provides limited information on the possible genetic abnormalities that embryos may have (8). Currently, there exists a great controversy on the optimal moment to carry out embryo transfer.
IN WHICH CASE DOES THIS TRANSFER USUALLY TAKE PLACE?
Day 2 transfer is usually indicated in cases of poorly responding patients. Indeed, it is also indicated when the sperm, oocyte and/or embryos are also of low quality and/or number (9, 10, 11).
WHAT DO EXPERTS SAY?
Several retrospective studies have compared embryo transfer on day 2, day 3 and day 5 after oocyte recovery, all of which presented conflicting results. A study performed by Mahdavi et al. among poor responder patients revealed no clinical differences between day-2 and -3 embryo transfer (10). However, this study found that pregnancy rates per oocyte retrieval and embryo transfer were significantly higher in the day-2 embryo transfer group compared to day 3 group. It is worth mentioning that other investigators did not find significant differences in pregnancy outcomes when they compared embryo transfer on day 2 and day 3 (11, 12).
Additional results from other studies have revealed higher clinical and ongoing pregnancy rates after embryo transfer on day 2 than on day 3 in poor responders. This suggests that the occurrence of miscarriage can be reduced by restricting embryo culture to only 2 days, which could also provide an alternative for managing poorly responding patients (11). That is the reason why embryo transfer on day 2 is still performed in many IVF centres; there is an actual risk of compromising the viability of embryos by prolonged in vitro culture in sub-optimal conditions, with an increased risk of obtaining no blastocysts to replace on day 5 (9, 13, 14).
Even though there seems to be a large number of benefits for these patients, certain disadvantages that may potentially occur must also be taken into account, as it can be seen below (Table 1).
PATIENTS IN WHICH TRANSFER D+3 SHOULD BE PERFORMED
There exists no criterion to select patients who should be transferred on D+3. Traditionally, embryo transfer has been performed on cleavage stage, so the chosen day was D+3 of embryo development (7). Generally speaking, embryo transfer was carried out on this day in all patients, until a culture medium was developed that allowed to keep embryos in the laboratory for 5-6 days, and with the exception of the cases previously mentioned (11).
SCIENTIFIC LITERATURE TO SUPPORT D+3 AS THE BEST DAY FOR EMBRYO TRANSFER
Many studies show contradictory results on whether it is better to transfer on D+2 or D+3. However, there are no significant differences as for implantation, clinical pregnancy or live birth rates when comparing transfer on these days. A study by Modares et al. (15) with patients under 40 years old showed a slight improvement in these results when transferring on D+3, although differences were not significant. The authors also showed embryo quality to be worse when the transfer was performed on D+3 than on D+2. Thus, implantation rate has been observed to be higher in D+3, because extending embryo culture for one day allows to discard those embryos that stop their development from D+2 to D+3 (16, 17).
Furthermore, it is necessary to consider that there are other external factors that affect embryo development and, consequently, the selection of the best day to transfer. Quinn et al. (18) determined that one of these factors is culture media. Thus, in sub-optimal lab conditions, it would be interesting to transfer on D+2 rather than D+3, in order to spend the shorter time possible in the media.
Regarding D+5 transfer, some studies have shown higher implantation rates in embryos transferred on the blastocyst stage compared to those transferred on D+3 (cleavage stage). However, it is necessary to consider that only 25% of embryos reach the blastocyst stage (15); this implies that the number of embryos transferred and vitrified in a cycle is lower than for D+2 and D+3. As a consequence, when considering cumulative pregnancy rates no significant differences are found between transferring on cleavage stage and blastocyst (7).
Again, benefits for the patients must be considered along with potential disadvantages (Table 2).
It has been observed that transfer on blastocyst stage helps to improve pregnancy rates and reduce the risk of a multiple pregnancy. Why? One reason might be that there is no method to determine whether embryos that initially seem to be of good quality are likely to develop up to blastocyst (19).
WHO ARE THE IDEAL PATIENTS?
1. Those with a large number of embryos (20).
2. Those whose day-3 embryos are of good quality (20).
3. Those in which day-1 embryos exhibit pro-nuclei and present a grading profile (20).
4. Young women with good ovarian response (21).
5. Those whose embryos display an early cleavage (22).
POTENTIAL BENEFITS OF BLASTOCYST-STAGE TRANSFER vs. CLEAVAGE-STAGE TRANSFER
First of all, the new culture media allow us to perform longer incubations in the laboratory, after which the best embryos can be selected with higher accuracy and with lower risk of aneuploidies (23). Moreover, there will exist a better synchronization between the embryo and the mother. Additionally, uterine contractility decreases during the luteal phase (24, 25). The size of these blastocysts is bigger, so some studies have found fewer cases of ectopic pregnancies in comparison to transfers on day 3 (26).
A parallel comparison of benefits vs. disadvantages for this procedure can be seen in below (Table 3).
IS IT BETTER TO TRANSFER ON DAY 5 OR ON DAY 6?
The optimal time for embryo transfer depends on a variety of factors, such as the embryo growth speed. However some studies have revealed both implantation and pregnancy rates to be more successful when embryos are transferred on day 5 compared to day 6. This is due to the fact that viability of embryos expanded on day 5 is higher than for those expanded on day 6 (30).
In conclusion, it seems difficult to define the most appropriate day for embryo transfer to be applied for each patient. Therefore, every single case must be individually analyzed. In addition, several factors should be taken into account when deciding on the day for embryo transfer, such as maternal age, sperm and oocyte quality or physiological response of the woman and/or the available embryos. By doing so, a good decision can always be made in order to improve implantation and pregnancy rates.
Authors: Shuyana Deba, Javier Del Río and Sara Sanz
Special collaboration: Álvaro Martínez Moro
Infertility affects millions of couples all around the world. In spite of the solutions to their problems reproductive technology can achieve, the efficacy is eventually limited by the number and the quality of the oocytes available from the woman. In actuality, such efficiency is determined by the ovarian reserve, the oocyte quality and the maternal age, among the most important factors (2).
Diminished ovarian reserve (DOR)
Since ovarian reserve defines the quantity and quality of the primordial follicle pool, diminished ovarian reserve (DOR) indicates a reduction in quantity in women of reproductive age. Consequently, it represents important cause of infertility in many couples. Moreover, DOR may be associated with low pregnancy rates and high pregnancy loss regardless of age, but further research is needed in order to fully understand its implications (3).
Advanced maternal age
It is well known that women’s fertility declines sharply after age 35 due to several factors, which include specific issues of reproductive organs (uterus and oviducts), general health and decreasing number and quality of oocytes over time. The oocyte pool starts to decline during foetal life and continues within the reproductive life of women. Oocyte quality also decreases as a consequence of the increased rate of aneuploidies observed with age: 74% at the age of 41–42, and up to 93% after the age of 42 (5). Advanced age is too associated with a reduction in the quality of the oocyte cytoplasm (ooplasm), which directly affects oocyte maturation (3).
What are the main reasons for this reduction in ooplasm quality? Mitochondria are one of the most important organelles, which are affected in different ways (6,7):
- Morphological and functional abnormalities
- Mitochondrial swelling
- Alterations in mitochondria's cristae
- Alterations of the membrane potential
- Alterations of the metabolic pathways in cummulus cells, which may result in impaired mitochondria biogenesis during oogenesis.
These effects are due to the higher ratio of mutation consequence of the proximity of these organelles to the respiratory chain, the inefficient repair mechanism and the exposure of histories. How these changes affect oocyte quality (8)? First of all, negative effects on chromosome segregation have been observed as a result of a decreasing ATP concentration (9,10). Additionally, defects have been found in different signalling pathways such as Ca2+ signalling, which affects fertilization and the subsequent embryo development (11).
Nevertheless, different mitochondrial haplogroups should be taken into consideration. These have different bioenergetic functions, including production of reactive oxygen species (ROS) and mitochondrial coupling efficiency, aspects that might affect the oocyte longevity (13). Consequently, new techniques are being developed in order to increase the reproductive options in women with oocyte problems. Recently, one of these techniques that have been highly treated in the media is the development of additional viable oocytes from polar body genomes (2).
HOW DOES TRANSFER OF POLAR BODY GENOME WORK?
Originally, the transfer of polar body has been applied to cases of infertility with a genetic cause, such as the presence of mitochondrial diseases. These cases can be treated with the use of donor oocytes in clinical practice. Additionally, another application is the formation of human metaphase II (MII) oocytes, which increases the number of available oocytes for an assisted reproduction cycle (2).
Two specific combined steps are needed. First, the donor oocyte spindle is removed, which requires the utilization of polarized light. Once located, it will be biopsied, obtaining an enucleated oocyte (14,15,16). Secondly, the patient polar body is biopsied, provided elimination of the spindle apparatus has been confirmed.
Once both processes have been performed, the last step is the introduction of the polar body genome inside the enucleated oocyte (17).
FUNCTIONAL HUMAN OOCYTES GENERATED BY TRANSFER OF POLAR BODY GENOMES
Hong Ma and his group have tried to test the efficiency and possible limitations of this technique (ref). The main objective to be achieved was the formation of spindles resembling those typical of MII oocytes, including the appropriate chromosome dosage.
HOW EFFICIENT IS THIS TECHNIQUE?
Although DAPI staining demonstrated that all polar body nuclear transfer (PBNT)-oocytes contained spindle-chromosome complexes, only two of five experimental oocytes formed metaphase spindles similar to intact MII oocytes. This low number may be due to residual meiotic activity in enucleated human MII oocytes, which is sometimes not enough to induce formation of normal MII-like spindles.
For a different cohort of oocytes, the rate of successful fertilization was 76%, still slightly lower than control oocytes. Furthermore, 42% of embryos reached blastocyst stage, indicating that most of the PBNT-oocytes were capable of completing the second meiotic division. Short tandem repeat (STR) analysis revealed that two sampled PBNT-blastocysts contained normal diploid chromosomes, determining that these embryos were completely viable.
WHAT CAN BE CONCLUDED?
• Polar body genome transfer seems to be a significant technique for the improvement of assisted reproductive technology (ART) outcomes and pregnancy rates, particularly for women with decreased ovarian reserve and low response to stimulation.
• The cytoplasm from young donor oocytes may reduce incidences of low cytoplasmic oocyte quality.
• It could provide an additional technique to support mitochondrial replacement therapy.
Nevertheless, this technique is not suitable for women who cannot produce mature oocytes, typical profile of ART patients. Additionally, incidences of aneuploidy resulting from errors in mitosis or in the second meiotic division may still occur because of women advanced age. Larger datasets from this technique are needed to confirm its efficacy and safety. Also, improving preimplantation genetic screening (PGS) is critical before eventual clinical application.
Authors: Shuyana Deba, Javier Del Río and Sara Sanz
Some weeks ago, Dr. Hayashi's group published a study in Nature in which they described how to generate functional mouse eggs from cultured stem cells. To better understand this experiment, it is necessary to explain the natural ovarian cycle. Although the experiment was carried out in mice, we believe these experiments could be of great importance in humans in the near future. Therefore, the following description applies to human ovarian cycle.
GONADAL DEVELOPMENT IN THE EMBRYO
As the yolk sac begins to incorporate into the embryo, germ cells migrate along the dorsal mesentery of the hindgut to the gonadal ridges, which they reach by the end of the fourth or early fifth week of development (2). Around week 7 of gestation, gonadal ridges undergo proliferation to form primitive sex cords, to which primordial germ cells (PGCs) are associated (Fig. 2). In the absence of both expression of the SRY gene product and male sex hormones, differentiation into female organs is determined (3,4). Whereas the origin of the follicular cells of the ovary remains unclear, likely candidates are cells from the coelomic epithelium and the mesonephros. The follicular cells eventually associate with the PGCs to form primordial ovarian follicles (3,4).
ORIGIN OF GERM CELLS
Human oocytes are derived from round PGCs that can be identified in the wall of the yolk sac as early as 24 days after fertilization. Once the gonad develops into an ovary, PGCs become oogonia, and mitotic divisions go on up to the seventh foetal month, and cease at some point shortly before birth (5).
By the 8-9th week after fertilization, some oogonia enter into prophase I of meiosis, thus becoming primary oocytes. Meiosis starts in the deepest part of ovary (medulla) and progresses towards the cortex. Some time after it has been incorporated into a primordial follicle, the oocyte arrests after diplotene, at a particular stage called dictyate or dictyotene. This means the oocyte enters into a long quiescence period that begins before birth and ends up either resuming after the LH surge (leading to ovulation) or in follicular atresia (3).
Even though the experiment by Hayashi's group has just been successfully performed in mice, the purpose of the investigation is eventually applying it to humans.
FOLLICULOGENESIS AFTER BIRTH
Once placental oestrogen disappears, gonadotrophin levels begin to increase after birth. This increment will last 12-24 months postpartum in women. Thus, the ovaries remain inactive until the beginning of pubertal stage, moment when they will start showing response to GnRH pulsatility (6). By this time, ovaries contains germ cells at the primordial follicle phase, which are composed of small immature oocytes arrested at dictyate, and surrounded by a single layer of flat squamous granulosa cells (7).
WHAT HAPPENS AFTER PUBERTY?
Once sexual maturity is reached, multiple primordial follicles are activated from their quiescent state, resuming folliculogenesis by cohorts every menstrual cycle. During follicle maturation, follicles are subjected to activin, AMH, FSH, inhibin, LH, TGFβ and a multitude of other molecules that control the process. At the beginning of this process, as the oocyte resumes meiosis follicular cells proliferate and surround the oocyte, which develops into a primary follicle (7,8,9).
Briefly, transition into the secondary or antral follicle stage involves the alignment of the stroma around the basal lamina and the development of an independent blood supply. Follicular cells of the underlying stroma, the granulosa cells, subsequently differentiate into an inner and an outer theca layers, the latter supporting this blood supply. The production of follicular fluid by these cells promotes the formation of the antrum in pre-antral follicles (10).
Progression through antral follicle development is promoted by FSH and coincides with (7):
- Completion of oocyte growth.
- Acquisition of competence to complete meiosis (dependent on fertilization).
- Continued granulosa cell proliferation.
- Differentiation of cummulus and mural granulosa cells upon formation of the follicular antrum.
Response to FSH causes growth of the follicle by proliferation of follicular cells and coalescence of the astral cavities, thus generating a follicle whose volume keeps increasing. The final stage of folliculogenesis takes place when the Graafian follicle reaches its maximum size (around 20 mm diameter). The LH surge triggers the ovulation of a metaphase II-arrested oocyte (along with the cummulus), capable of being fertilized and able to support embryonic development (7). Although there are notable differences between species, in humans, usually only one follicle is capable of completing growth and maturation up to ovulation (preovulatory), due to its more efficient response to FSH in a "competitive manner" against the rest of developing follicles. The one follicle will progress until subsequent fertilization (dominant follicle), whereas the remaining follicles in the cohort will undergo atresia (8).
ABOUT PLURIPOTENT STEM CELLS
For centuries, scientists have investigated how oocytes and sperm develop in mammals. Most of this work has been performed in non-human species, mainly the mouse. Human pluripotent stem cells (PSCs)-derived gamete research presents notable scientific value and potential, both for understanding basic mechanisms of gamete biology and for overcoming clinical problems (11).
PSCs are currently used to answer important scientific questions such as the role of specific genes in early germ cell development, involved pathways, interaction between germ cells and supporting somatic cells, or partly or entirely in vitro PSCs-derived eggs and sperm (11).
Some of the potential benefits of these new techniques could be (11):
- Additional options for assisted human reproduction.
- New ways to prevent and treat infertility, genetic diseases and some types of cancer.
- Optimization of the number of oocytes obtained from women.
"IN VITRO RECONSTITUTION OF THE ENTIRE CYCLE OF THE MOUSE FEMALE GERMLINE" (1)
The authors have shown a method to reconstruct the entire process of mouse oogenesis in vitro, using embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in order to obtain fully functional and mature oocytes. Why does this study mean so much?
First of all, this is a big achievement in the field of regenerative medicine, through which it is possible to study the function of certain genes involved in oogenesis. Furthermore, it sets the ground for future similar studies and culture systems in other specie, and as such, it may result in a great advance in reproductive medicine.
However, which are the limitations for this approach?
The culture system requires somatic cells, which are obtained from embryos. So far, this is an ethical and legal issue in humans. A feasible solution would be to obtain the gonadal somatic cell-like cells from PSCs. The authors found that the differentially expressed genes (DEGs) were down-regulated in in vitro MII oocytes, whereas in vivo MII oocytes showed an increase in the number of transcripts. This maybe a consequence to the fact that, during in vitro growth (IVG) and in vitro maturation (IVM), oocyte development was jeopardized within a subset of (or perhaps all) the oocytes. Why? Misregulation of such genes, related to mitochondrial functions, might attenuate the potential of the in vitro MII oocytes. This problem could be solved by an analysis of the metabolic pathway, which could in turn provide information to refine both IVG and IVM.
RESULTS, "PROBLEMS" AND "ACHIEVEMENTS" OF THE EXPERIMENTS (1)
As it can be seen concluded from above, it seems clear that further tests are still needed, which would help measure some aspects of the viability and functionality of this in vitro generated gametes.
1. Hikabe O, Hamazaki N, Nagamatsu G, Obata Y, Hirao Y, Hamada N et al. Reconstitution in vitro of the entire cycle of the mouse female germ line. Nature. 2016;.
2. Baillet A, Mandon-Pepin B. Mammalian ovary differentiation–A focus on female meiosis. Molecular and Cellular Endocrinology. 2012;356(1-2):13-23.
3. Bolender DL, Kaplan S. Chapter 3 – Basic Embryology. In: Fetal and Neonatal Physiology. 2017. p. 23–39.e2.
4. Parker KL, Schimmer BP. Chapter 8 – Embryology and Genetics of the Mammalian Gonads and Ducts. In: Knobil and Neill’s Physiology of Reproduction. 2006. p. 313–36.
5. Racowsky C, Gelety TJ. Chapter 4 – The Biology of the ovary. Princ Med Biol. 1998;12:77–102.
6. David Vantman B, Margarita Vega B. Fisiología reproductiva y cambios evolutivos con la edad de la mujer. Revista Médica Clínica Las Condes. 2010;21(3):348-362.
7. Hutt K, Albertini D. An oocentric view of folliculogenesis and embryogenesis. Reproductive BioMedicine Online. 2007;14(6):758-764.
8. Green L, Shikanov A. In vitro culture methods of preantral follicles. Theriogenology. 2016;86(1):229-238.
9. Palermo R. Differential actions of FSH and LH during folliculogenesis. Reproductive BioMedicine Online. 2007;15(3):326-337.
10. Gougeon A. Human ovarian follicular development: From activation of resting follicles to preovulatory maturation. Annales d'Endocrinologie. 2010;71(3):132-143.
11. Mathews D, Donovan P, Harris J, Lovell-Badge R, Savulescu J, Faden R. Pluripotent Stem Cell-Derived Gametes: Truth and (Potential) Consequences. Cell Stem Cell. 2009;5(1):11-14.
Authors: Javier Del Río and Sara Sanz
Genetic problems in the embryo are one of the most important causes of pregnancy loss and miscarriage. However, identifying embryo mosaicism as the cause of genetic problems during development is not an easy task.
WHAT IS A MOSAIC EMBRYO?
The term mosaicism refers to the presence of more than just one cell line, which present different chromosome count (1). Additionally, the most common situation in these cases is the presence of a mixture of distinct aneuploid cells, rather than of a variety between euploid and aneuploid cells. These are the embryos that may be at risk of misdiagnosis (2).
There are four possible types of mosaic embryos (3,4):
1. Embryos with a mix of euploid and aneuploid cells in the trophectoderm (TE), and with aneuploid cells in the inner cell mass (ICM).
2. Embryos with a mix of euploid and aneuploid TE cells and euploid ICM.
3. Embryos with euploid TE cells and aneuploid ICM.
4. Embryos with aneuploid TE cells and euploid ICM.
Even though there exists no specific cut-off to determine mosaicism, the Preimplantational Genetic Diagnosis International Society (PGDIS) suggests an embryo with more than 20% of aneuploid cells to be considered as mosaic. This means lower levels of mosaicism should be treated as normal (euploid) (5).
It has been traditionally thought that only genetic problems in the oocyte or the sperm could be responsible for embryo mosaicism. Nevertheless, it is currently postulated that this also occurs during the first mitotic divisions, when maternal transcripts control the cell cycle of the early embryo (6).
NORMAL INDICATIONS FOR CHROMOSOMAL TESTING TO DETECT EMBRYO MOSAICISM
1. Advanced maternal age. This is the most common cause for aneuploid problems (7). A recent study has shown that women between the ages of 35 and 43 years have more probabilities (an increase ranging from 28 to 78%) of presenting mis-segregation for the most clinically relevant aneuploidies, namely chromosomes 13,16,18,21 and 22 (8).
2. Severe male factor infertility. Even though levels of sperm aneuploidy are associated with increased levels of chromosomal abnormalities in embryos (9), such abnormalities could also arise from certain males who do not present any chromosomal abnormality a priori. Such could be specific cases of oligoastenozoospermic patients (10).
3. Recurrent implantation failure (RIF). In spite of the lack of specifications for such diagnosis, it is usually defined as the occurrence of three or more failed IVF attempts due to an unidentified cause. RIF is the usual diagnosis in those cases in which after a cumulative transfer of more than 10 good-quality embryos, the eventual result is IVF failure (7,11,12,13).
4. Recurrent miscarriage. The definition of this concept may vary for every country. However, generally speaking it can be defined as the occurrence of 3 or more consecutive miscarriages once pregnancy has reached at least 14 weeks (14). The main cause for this problem seems to be aneuploidy, which has been identified as the leading cause in a high percentage of miscarriages (15,16).
5. Previous trisomic pregnancy. Cases in which there has been a previous trisomic pregnancy entail higher probability of suffering from another aneuploid conception. Therefore, it is in this group of patients in which it would be beneficial to conduct a study to find out possible related causes (17).
BIOPSY TECHNIQUES TO STUDY A CASE OF MOSAICISM
Although there are different biopsy strategies, depending on the embryo stage, blastocyst biopsy is recommended over both polar body and blastomere biopsies in those cases in which mosaicism is suspected. Blastocyst biopsy is less invasive, it is possible to extract a higher number of cells, which increases the probability to confirm mosaicism, and it is cheaper than the other techniques due to the lower number of embryos required for biopsy (7). Furthermore, if cells with different chromosome complements are widely distributed throughout the trophectoderm, there might be a good chance of capturing a representative sample. If cells are clustered, mosaicism could not be easily detected, thus providing false normal results (18).
TECHNIQUES EMPLOYED TO DIAGNOSE MOSAICISM
The earliest trials of PGD involved the use of karyotyping and PCR. By the mid-90s, the use of cytogenetic techniques such as FISH allowed for the progress of preimplantation diagnosis. This very own approach was later shown to impose important technical limitations to the analysis, and so it was encouraged the development of new technologies that could minimize the errors in diagnosis (19).
Fluorescence in situ hybridization (FISH)
It allows for the analysis and identification of chromosomes or chromosome fragments with 5-10 fluorescently labelled molecular probes from one cell (blastomere) (Fig. 2). This cell can be biopsied from day-3 embryos, it could belong to the trophectoderm from blastocysts or it could also be a polar body biopsied from an oocyte or a zygote. Therefore, PGS–FISH diagnosis is limited to the most common abnormalities involving chromosomes 13, 15–18, 21, 22, X and Y. Some studies of preimplantation embryos diagnosed by using this technique estimate a 5-7% error caused by mosaicism when embryos are reanalyzed. Additionally, FISH is being rapidly replaced by other DNA analysis methods with higher efficiency (20,21).
Array Comparative Genomic Hybridization (aCGH)
This technique relies on whole genome amplification from one or more blastomeres (Fig. 3). It provides a quantitative analysis based on the comparison between the relative amount of tested DNA and the control DNA. Thus, chromosome imbalances such as aneuploidies, unbalanced translocations, deletions and duplications are easily detected. However, since balanced chromosome rearrangements such as reciprocal translocations or inversions do not affect copy number, such alterations cannot be identified (19,20). When blastomeres are analyzed by aCGH, the error rate measured is merely 2% (21,23). Notably, some studies have found that PGS-aCGH after blastocyst biopsy provides higher implantation and pregnancy rates than PGS-FISH (24).
Next Generation Sequencing (NGS)
Next Generation Sequencing belongs to the group of Massively Parallel Sequencing (MPS) methods that allow for parallel processing of an extremely large number of nucleic acid molecules (Fig. 4). As a result of sequencing on a microspace scale, it has been possible to drastically increase the amount of information collected during one test up to an entire human genome. Also, it is the only method that allows for analysis of all chromosomes (aneuploidies or translocations) and mutations responsible for any single-gene disease, just using one biopsy and in a single step (20). Although clinical results have documented high pregnancy rates following transfer of screened embryos, further data along with an extended use in clinical application are required to better define the role of NGS in PGS. Nevertheless, it seems that this method may actually lead to reduced costs per patient, thus allowing IVF couples a wider use of PGS for choosing the most competent embryo for transfer (26).
DIFFICULTIES WHEN MANAGING RESULTS
The detection of mosaicism at an early stage does not mean that it will spread along embryo development (27). However, the utilization of chromosome identification techniques as part of the IVF process makes it possible to identify embryos “at risk of mosaicism’’ in order to select those that are suitable for transfer (18).
Mosaic embryos are supposedly less competent than others due to a reduced implantation potential. Therefore, by discarding mosaic embryos implantation rates should be improved and, simultaneously, embryo loss rates reduced. Nevertheless, mosaic embryos may still have reproductive potential, and consequently they could still be viable. Furthermore, discarding embryos capable of producing healthy children will decrease pregnancy rates in those patients who get a low number of blastocysts in the pool of transferable embryos (18).
It is important to take into account the reaction of the patients when they are informed about their embryos being at risk for mosaicism, what may entail genetic abnormalities, reduce implantation rates, increase loss risk and even diminish obstetrical and neonatal outcomes. However, there is not a simple answer when patients decide to transfer a mosaic embryo; either way, the obstetrical team should be informed for future screens (18).
SUGGESTED GUIDELINES BY THE PREIMPLANTATINO GENETIC DIAGNOSIS INTERNATIONAL SOCIETY (PGDIS)
This article has been selected for publication in the Scientists in Reproductive Technologies (SIRT) Newsletter of The Fertility Society of Australia: DEL RÍO, J. and SANZ, S. (2017) Mosaic embryos are capable of producing healthy children. How to handle it? Fertility Society of Australia - SIRT Newsletter 4(20): 12-15.
Authors: Javier Del Río and Sara Sanz
Although most of the genetic material of eukaryotic cells is located inside the nucleus, mitochondria are organelles that also possess a certain amount of DNA. Mutations in mitochondrial DNA (mtDNA) or nuclear genes involved in mitochondrial function are cause of infertility and diseases, not only in individuals, but also in their offspring. In these cases, one of the solutions known to be efficient in order to conceive and give birth to a healthy child is the "three-parent in vitro fertilization" approach.
THE MITOCHONDRIAL GENOME IS MATERNALLY INHERITED
During fertilization, mitochondria from sperm are normally eliminated by a ubiquitin-dependent mechanism. As a consequence, in case the father carries the mutation both his health and fertility could become affected, but never his offspring (2). By contrast, mitochondria in the oocyte must present a specific location and distribution pattern (which represents an actual sign of oocyte maturation), and they are solely inherited from the mother.
WHY ARE MUTATIONS IN mtDNA OR IN NUCLEAR GENES INVOLVED IN MITOCHONDRIAL FUNCTIONS SO PROBLEMATIC?
Mitochondria provide energy to the cells through oxidative phosphorylation, and so mutations in their genome mainly affect structures form the nervous system, heart, skeletal muscle, pancreas, gonads, colon, blood, kidney or liver (3). Why these structures? The higher the energy demand is, the higher the need for more mitochondria in the cells (4). Also, cells that present a slower division process are more likely to present some kind of mtDNA mutation (5).
HOLOPLASMY vs. HETEROPLASMY
The situation in which all cells from an individual contain identical mtDNA (mutated or otherwise) is known as holoplasmy. By contrast, heteroplasmy is defined as the condition in which part of the mitochondria from the same individual present a DNA content that is different from the other. These cases are the most common among patients affected by mitochondrial DNA diseases (6).
WHY DOES HETEROPLASTY REPRESENT A PROBLEM?
Even though heteroplasmy implies the presence of two different DNA contents in the cell, cells with great amounts of mutant (or affected by a specific condition) mitochondria respond to proliferate their entire DNA. This is why the percentage of mutant/affected mtDNA tend to increase in certain tissues (7).
THE "BOTTLENECK EFFECT" AND THE "THRESHOLD EFFECT"
During oogenesis, only a subset of molecules of mtDNA are eventually amplified and passed on to the offspring (8). This effect explains why it is possible to obtain homoplasmic individuals in just a few generations (2).
Previous reports on human diseases caused by an mtDNA mutation have shown that the mutation needs to be present at a certain percentage in order to manifest pathological effects. Typically, this percentage should be higher than 60-80% (8,9), although it also depends on age, affected tissues, type of mutations, etc. (4)
WHY TO APPLY THE "THREE-PARENT IN VITRO FERTILIZATION" APPROACH?
It might be reasonable to think of other possibilities to treat patients suffering from mitochondrial diseases in order to achieve pregnancy. Rather than prenatal diagnosis or preimplantation genetic diagnosis, the "three-parent in vitro fertilization" technique because (4):
In the first case:
1. It needs a uniform mtDNA distribution in the extra-embryonic and foetal tissues.
2. It needs the mutant DNA load to remain constant over time.
3. There must be a close relation between the severity of the disease and the amount of mutant DNA.
As for preimplantation genetic diagnosis (6,9):
1. It is not applicable to patients with high levels of heteroplasmy.
2. It reduces but does not eliminate the risk of suffering from a mitochondrial disease-related condition.
3. The amount of tDNA found in blastomeres or the trophectoderm does not represent the whole embryo.
4. This approach is not an efficient diagnosis due to the combination of heteroplasmy and the "bottleneck effect".
KNOWB TECHNIQUES TO BE USED FOR MITOCHONDRIAL REPLACEMENT
It involves the transfer of the two pronuclei from a zygote affected by diseased (or mutated) mitochondria into an enucleated zygote containing healthy mitochondria. Even though this technique has not yet been performed in humans, the efficiency of pronuclear transfer in mice has been adversely affected by descendants bearing high levels of carryover mtDNA (10).
Polar body transfer
Since the polar body has a lower proportion of mitochondria around it, this is currently considered the best method for preventing the transmission of mutated mtDNA on to the next generation (9,10). Embryos derived from polar body transfer support normal fertilization and are capable of producing live offspring in mouse. Polar body transfers leading to a minimal amount of affected mtDNA carryover have demonstrated the great potential of this technique for preventing inherited mitochondrial DNA diseases (9,10).
When applied in mice, this technique has shown the best success rate so far due to the transfer of mitochondria being lowered to a minimum (9).
This technique involves transferring the meiotic spindle along with the associated chromosomes, the spindle-chromosome complex (SCC), from an unfertilized oocyte with affected mitochondria into an enucleated healthy mitochondria-containing oocyte (11). This technique recently became popular when performed by Dr. John Zhang and his team, hitting the media within the latest weeks. However, potential problems could arise; just as for the previous techniques, the spindle is also surrounded by mitochondria, and so they could too be introduced into the ooplasm, thus causing heteroplasmy (12).
WHAT DOES LAW STATE REGARDING MITOCHONDRIAL REPLACEMENT?
So far, scientific societies are very skeptical about experimental techniques. Thus, this particular approach is specifically prohibited by the Food and Drug Administration (FDA) in the US. It can only be performed in countries such as Mexico, where legislation is more flexible, or in UK, where it was approved for application in very specific cases (14).
CURRENT DATA ON MITOCHONDRIAL REPLACEMENT
As it has been previously mentioned, cases of cytoplasm transfer have been performed. In fact, there have been around 30-50 live births from this technique. However, newborns presented certain genetic defects, and so this technique was banned and replaced by pronuclear transfer and meiotic spindle transfer (14). Such data demonstrate the potential damage that could be inflicted to the embryo when performing these techniques (12,15). In addition to this, ethical issues must also be taken into account, which means the sole possibility of successfully applying a specific procedure does not imply its moral appropriateness. In order to guarantee so, a committee of experts should pronounce their opinions and reach a consensus about it. On a related note, long-term effects derived from these procedures are still unknown, and so it would be necessary to monitor all babies born through these techniques.
This post has been published in the Scientists in Reproductive Technologies (SIRT) newsletter, a special interest group representing the scientific membership of The Fertility Society of Australia.
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