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Year : 2021  |  Volume : 14  |  Issue : 5  |  Page : 3-30
 

ISAR consensus guidelines on add-ons treatment in in vitro fertilization


1 Managing Director, Rainbow IVF, Agra, Uttar Pradesh, President ISAR (2019), India
2 MBBS, MCE, Chief Embryologist & Director-Rainbow IVF, Agra (Uttar Pradesh), India
3 M.Sc., Laboratory Director, Bloom IVF Group, Mumbai, India
4 Chief Embryologist, Janini IVF, New Delhi, India
5 Scentific Head and Consultant Embryologist Ferty Fertility Center, Secunderabad, Telangana, India
6 Senior Embryologist, Coopersurgical India Pvt Ltd, Mumbai, Maharashtra, India
7 Director and Chief Embryologist, Elixir Fertility Centre, Delhi, India
8 PhD, Consultant, Cooper Surgicals, Scientific Consultant, Trivector Biomed, Mumbai, India
9 Director-IVF lab., Chief Embryologist, Akanksha Hospital and Research Institute, Anand, Gujarat; Lab Director - ZIVYA IVF, Mumbai, Maharashtra; Secretory -SKHPL Institutional Ethics Committee, Anand, Gujarat, India
10 Lab Director, Mamta Fertility Hospital, Hyderabad, India
11 Senior Scientist, Department of Women and Children's Health, Karolinska Institute, Stockholm, Sweden
12 Senior Embryologist at Lilavati Hospital and Research Center (IVF Department), Mumbai, Maharashtra, India
13 Director, Institute of Human Reproduction, Guwahati, India
14 Director - IVF Lab, Akanksha IVF Center New Delhi, India

Date of Web Publication16-Nov-2021

Correspondence Address:
Dr. Keshav Malhotra
MBBS, MCE, Chief Embryologist & Director-Rainbow IVF, Agra (Uttar Pradesh)
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0974-1208.330501

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   Abstract 


Study Question: What are the good practices for the use of ADD-ON Treatments in IVF cycles in INDIA? What is Already Known: Add on treatments in IVF are procedures and technologies which are offered to patients in hope of improving the success rates. A lot of add on treatments exist; most of them have limited evidence and data for the Indian patient population is miniscule. These interventions may have limited effects, so it is imperative that any new technology that is offered is evaluated properly and has enough evidence to suggest that it is safe and effective. Study Design, Size, Duration: This is the report of a 2-day consensus meeting where two moderators were assigned to a group of experts to collate information on Add on treatments in IVF in INDIA. This meeting utilised surveys, available scientific evidence and personal laboratory experience into various presentations by experts on pre-decided specific topics. Participants/Materials, Setting, Methods: Expert professionals from ISAR representing clinical and embryology fields. Main Results and the Role of Chance: The report is divided in various components including the health of the Offspring, the various ADD ons available to an ART center, consensus points for each technology & qualifications and trainings for embryologists, the report and recommendations of the expert panel reflect the discussion on each of the topics and try to lay down good practice points for labs to follow. Limitations, Reasons for Caution: The recommendations are solely based on expert opinion. Future availability of data may warrant an update of the same. Wider Implications of the Findings: These guidelines can help labs across the country to standardise their ART services and improve clinical outcomes, it will also motivate clinics to collect data and report the use of Add ons to the national registry. Study Funding/Competing Interest(S): The consensus meeting and writing of the paper was supported by funds from CooperSurgical India.



How to cite this article:
Malhotra J, Malhotra K, Kamat S, Mishra A, Chatterjee C, Nair S, Ghosh P, Mehta R, Bhadraka H, Srinivas S, Kumar L, Mistry R, Goenka D, Kant G. ISAR consensus guidelines on add-ons treatment in in vitro fertilization. J Hum Reprod Sci 2021;14, Suppl S1:3-30

How to cite this URL:
Malhotra J, Malhotra K, Kamat S, Mishra A, Chatterjee C, Nair S, Ghosh P, Mehta R, Bhadraka H, Srinivas S, Kumar L, Mistry R, Goenka D, Kant G. ISAR consensus guidelines on add-ons treatment in in vitro fertilization. J Hum Reprod Sci [serial online] 2021 [cited 2022 May 26];14, Suppl S1:3-30. Available from: https://www.jhrsonline.org/text.asp?2021/14/5/3/330501





   Introduction to Add-Ons Treatment in In vitro Fertilization Top


Add-ons are classified as optional treatments that can be offered with standard fertility treatments at an additional cost. Some techniques may have shown promising results in the initial studies, or they may have been around for several years but have not yet proven to improve pregnancy or birth rates.[1]

These guidelines are designed primarily as an educational resource for centers offering add-on treatments to help them in providing evidence-based medical services.[1]


   The Health of the Offspring Top


Background

Over the years, there have been concerns about the short-term and long-term risks for children conceived through assisted reproductive technology (ART) compared to naturally conceived children. Children conceived through ART have been thought to be phenotypically and biochemically different from those who conceived naturally, but the mechanisms underlying these differences and the subsequent health implications are unclear.[2] Health parameters that need to be monitored are namely birth defects, growth and developmental delays, blood pressure and blood glucose levels, predisposition to asthma, neurological dysfunction, autism, mental disorders, cancers, retinoblastoma, retinopathy, de novo imprinting disorders, and microdeletions.[3],[4]

Supporting evidence



Consensus on the health of children conceived by assisted reproductive technology



Recommendations

  • Counsel the patients for the associated risks[9]
  • Take informed consent whenever necessary.





   DNA Fragmentation Index, Computer-Assisted Semen Analysis, and Sperm Quality Analyzer Top


Background

DNA fragmentation index (DFI) can be tested using the laboratory investigations such as terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), COMET, sperm chromatin structure assay (SCSA), and sperm chromatin dispersion tests [Table 1]. However, some of these tests have limitations such as interlaboratory and interobserver variability. The cutoffs for many tests are not described clearly. Of the mentioned tests, SCSA and TUNEL are used by most of the clinicians as they are worldwide standardized and validated tests for all users.[10],[11],[12],[13],[14],[15]
Table 1: Advantages and disadvantages of the technologies

Click here to view


Supporting evidence on factors affecting DNA fragmentation index

Age and DNA fragmentation index

Increasing male age is significantly associated with higher DFI. The impact of increasing male age on DFI revealed that males >45 years of age had the highest DFI and the lowest high DNA stainability compared to all other age groups (P < 0.001). When intrauterine insemination (IUI)–artificial insemination (AIH) pregnancy rates were compared between patients with DFI <30 vs. >30, the pregnancy rates achieved were 14% and 3%, respectively. Serial ejaculation every 24 h for 4 days showed a reduction in DNA fragmentation.

Performing a DFI test in unexplained infertility before planning an IUI is recommended because there is a strong negative correlation between DFI and time to pregnancy with IUI. An early intracytoplasmic sperm injection (ICSI) based on the DFI test prevents the loss of valuable biological time, increases cost of failed IUI cycles of repeated unsuccessful IUI, and decreases the time to pregnancy.

Lifestyle factors and DNA fragmentation index

Lifestyle factors such as smoking, obesity, drugs, diabetes, seminal infection, environmental/occupational exposures, and clinical varicocele can have a negative impact on sperm quality. Sperm DNA testing can help reinforce the importance of lifestyle modification and antioxidant therapy and monitor the patient's response to an intervention.

Several studies have been conducted to observe the impact of DFI and its effect on ICSI outcomes which have been further correlated with lifestyle factors (age, body mass index [BMI], and smoking). In summary, the results of this study indicated that 14% of men had high DNA fragmentation. BMI and smoking showed a positive correlation with increased DFI.[16]

Iatrogenic factors and DNA fragmentation index
Table 2: Iatrogenic causes of DNA fragmentation index

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Sperm processing and DNA fragmentation index

The mean DFI of the sample after processing (swim-up, density gradient centrifugation, or density gradient followed by swim-up) was significantly lower when compared with fresh and simple wash samples.

Conclusion

  • The current semen analysis testing is incomplete to assess male fertility
  • Sperm DNA fragmentation testing has strong associations with every fertility checkpoint in ART
  • Sperm DNA fragmentation has strong associations with miscarriage rate
  • Sperm DNA fragmentation testing can explain “unexplained” infertility.


Consensus



Consensus recommendations




   Sperm Selection Techniques Top


Background

In natural conception, a few sperm cells reach the ampulla or the site of fertilization. This population is a selected group of cells since only motile cells can pass through cervical mucus and gain initial entry into the female reproductive tract.

Natural sperm selection in the humans is a rigorous process, resulting in the highest quality sperm reaching and having an opportunity to fertilize the oocyte. Relative to other mammalian species, the human ejaculate consists of a heterogeneous pool of sperm, varying in characteristics such as shape, size, and motility. The sperm selection techniques provide highly motile sperm populations or try to replicate the complex selection processes as seen in nature. Several methods have now been developed to mimic some of the natural selection processes that exist in the female reproductive tract.[17]

Advanced sperm selection techniques have developed as means of improving ART outcomes in specific clinical scenarios. Any selection mechanisms taking place in vitro must not harm the spermatozoa or modulate their interaction with the female reproductive tract if fertility is to be maintained. Biomimetic selection of the best quality spermatozoa for artificial insemination (AI) or for cryopreservation could improve pregnancy rates and may help reverse the decline in fertility.[18]

New technologies are being developed to aid in the diagnosis, preparation, and selection of spermatozoa in ART.

There are different techniques involved in the sperm selection process, namely direct swim-up, pellet swim-up, density gradient, physiological intracytoplasmic sperm injection (PICSI), intracytoplasmic morphologically selected sperm injection (IMSI), MACS, microfluidics, sperm birefringence, and others, advantages and disadvantages of which are discussed below:
Table 3: Sperm selection techniques: Advantages and disadvantages

Click here to view


Importance of sperm selection

We need to assume that treatments are still far from being 100% effective, as many patients fail to achieve pregnancy and others frequently need several attempts to achieve parenthood. Even when using the best gametes, obtained from young, healthy, and previously fertile donors, it can take several embryo transfers to achieve a pregnancy in certain patients. In relation to the egg, it has been reported that <7% of the retrieved oocytes lead to live birth. From the male viewpoint, the improvements needed are closely linked with the need to establish robust sperm quality indicators, to use them as a diagnostic tool to increase the success of ART, and to design appropriate strategies for sperm selection. Implementing such sperm diagnostic and/or selection techniques could significantly improve live birth rates, still acknowledging that outcomes will also depend heavily on oocyte and endometrium quality.[10]


   Surgical Sperm Retrieval Laboratory Requirements and Protocols Top


Background

Different surgical methods such as percutaneous epididymal sperm aspiration (PESA), MESA, testicular sperm aspiration (TESA), testicular sperm extraction (TESE), and micro-TESE have been developed to retrieve spermatozoa from either the epididymis or the testis according to the type of azoospermia [Table 4], i.e., obstructive or nonobstructive.[19] Laboratory techniques are used to remove contaminants, cellular debris, and red blood cells following collection of the epididymal fluid or testicular tissue. Surgically-retrieved spermatozoa may be used for ICSI and/or cryopreservation.
Table 4: Advantages and disadvantages of sperm retrieval techniques for assisted reproduction[20]

Click here to view


Conclusion

ICSI provides lower fertilization rates per injected oocyte as well as clinical pregnancy and delivery rates when testicular spermatozoa from men with NOA are used in comparison to ejaculated sperm or epididymal/testicular sperm from men with OA. Testicular spermatozoa from men with severely impaired spermatogenesis could have a higher tendency to carry deficiencies such as the ones related to the centrioles and genetic material, which could ultimately affect the capability of the male gamete to activate the egg and trigger the formation and development of a normal zygote and a viable embryo. The risks of congenital malformations, infertility, and other diseases in children conceived are still poorly determined.

The laboratory plays a crucial role in finding sperms and selection of the best-quality sperm for ICSI. Laboratory personnel should adhere to strict procedures for better clinical outcomes, which are listed below:

  • Receive the best-quality surgically retrieved specimen possible, with minimal or no contaminants such as red blood cells and microorganisms
  • Minimize iatrogenic cellular damage during sperm processing by mastering the technical skills and controlling factors such as centrifugation force, duration, temperature variation, laboratory air quality, dilution and washing steps, quality of reagents, culture media, and disposable materials
  • Improve sperm fertilizing potential, if possible by selecting viable sperm for ICSI when only immotile spermatozoa are available.


In vitro maturation: Background

Oocyte in vitro maturation (IVM) is a process in which oocytes are retrieved from the antral follicles of unstimulated or minimally stimulated ovaries and matured in vitro. IVM of human oocytes has emerged as a promising procedure. This technology has advantages over controlled ovarian stimulation, such as reduction of costs, simplicity, and elimination of ovarian hyperstimulation syndrome (OHSS). By elimination or reduction of gonadotropin stimulation, IVM offers eligible infertile couples a safe and convenient form of treatment, and IVM outcomes are currently comparable in safety to those of conventional IVF. IVM has been applied mainly in patients with polycystic ovary syndrome or ultrasound-only polycystic ovaries.[15]

In vitro maturation of oocytes: Indications

Marked improvements in the clinical and laboratory aspects of IVM treatment have led to better clinical outcomes. The recent development of fertility preservation has extended the indications of IVM beyond patients at risk of OHSS. IVM may represent a viable option in patients suffering from ovarian resistance to FSH, whose ovaries are endowed with an average number of antral follicles. Because IVM may be performed without exogenous gonadotropin administration, therefore avoiding supraphysiologic serum estradiol (E2) levels, it is applicable in emergency situations as well as in patients suffering from E2-sensitive diseases. Given the simplicity and the safety of the IVM procedure and the possibility to combine immature oocyte retrieval with ovarian cortex cryopreservation and given the risk of disease recurrences after grafting in some cases, we consider that IVM should systematically be offered to candidates undergoing ovarian tissue freezing. In leukemia patients, IVM currently represents the only viable option of fertility preservation as transplantation of cryopreserved ovarian tissue is still contraindicated.[21]



Traditional in vitro fertilization versus in vitro maturation



In vitro maturation: Advantages and disadvantages



Recommendation

In accordance to the Practice Committees of the American Society for Reproductive Medicine and the Society for Assisted Reproductive Technology, the following recommendations are suggested:



Artificial oocyte activation

Indications of artificial oocyte activation

Artificial oocyte activation (AOA) is mainly used in case of oocyte-related activation deficiency, IVM oocytes, low numbers of oocytes at retrieval, severe teratozoospermia, severe oligoasthenoteratozoospermia (OAT), globozoospermia, surgical sperm collection either for azoospermia or high sperm DNA fragmentation index, previous fertilization failure or poor fertilization, unexplained infertility, and frozen-thawed oocytes.

Assisted oocyte activation is not beneficial for all patients with a suspected oocyte-related activation deficiency

Despite the success of ICSI, total fertilization failure (TFF) still occurs in 1%–3% of all ICSI cycles. ICSI followed by AOA (ICSI-AOA) can restore fertilization, most efficiently in cases of sperm-related fertilization deficiency. The indication for ICSI-AOA is less apparent when the capacity of the sperm to activate oocytes is considered normal, as proved by a heterologous ICSI model, such as the mouse OAT.

According to Vanden Meerschaut et al., for patients with a suspected oocyte-related activation deficiency, as diagnosed by a heterologous ICSI model, the indication for ICSI-AOA remains debatable.[23]

The study data showed that ICSI-AOA is very efficient in patients with a suspected oocyte-related activation deficiency and previous TFF after conventional ICSI. In contrast, when there was a history of low fertilization (LF) in another center, one should be careful and test the efficiency of ICSI-AOA on half of the sibling oocytes, because ICSI-AOA is not always beneficial for patients with previous LF and a suspected oocyte-related activation deficiency. For these patients, a split ICSI-AOA cycle using sibling oocytes can help distinguish between a molecular oocyte-related activation deficiency and a previous technical or other biological failure. Moreover, this split ICSI-AOA strategy enables us to set the appropriate plan for future treatment cycles.

Calcium ionophore method for artificial oocyte activation

Calcium ionophore method for AOA has the most reports, and the resulting children have no reports of anomalies. Clinical use of these agents in assisted reproduction is limited by insufficient knowledge about their potential cytotoxic, teratogenic, epigenetic, and mutagenic effects on oocytes and embryos. It should be noted that issues of genetic safety and abnormal imprinting have not been addressed for the combined use of these oocyte activation methods. However, some studies revealed no physical or mental developmental disorders associated with babies born through these procedures, even 12 months after birth.


   Culture Media Additives: Embryo Top


Background

For a successful pregnancy to occur, embryos that can implant are essential. Several studies have suggested that the composition of the media that embryos are cultured in may have an impact on the quality of embryos generated in IVF/ICSI cycles, thereby influencing implantation and pregnancy rates.[24]

In the past, the culture of human gametes and embryos has been performed with surprisingly little standardization and a lot of experimentation. Today, commercially available culture media range from simple 8–10 different salts and sugars to media containing nearly 80 various components including amino acids, lipids, vitamins, trace ions, and bioactive molecules, such as hormones and expression modulators. Today, culture media for embryos can be used either as a sequential system, with different compositions for days 0–3 and 3–6, or as a single medium used for the whole culture period.[25]

Various culture media have different effects on the epigenome of the developing pre-embryo, and data suggest that the in vitro culture of gametes and embryos may influence the phenotype of the offspring.

[Table 5] summarizes the currently available knowledge on the main components of culture media and their possible effects on embryo and perinatal development and long-term outcomes.[26]
Table 5: Evidence-based review on culture media Culture media Evidence-based review additives

Click here to view


Implantation-promoting medium

Hyaluronan-rich media

Hyaluronan-enriched embryo transfer (ET) medium aids in implantation of embryos and hence improves pregnancy rates in IVF-ET cycles (IVF-ET). In patients with recurrent implantation failure, it may be considered as a useful transfer medium.[35]

Effect of granulocyte-macrophage colony-stimulating factor in embryo culture medium

Ziebe et al. established that the addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) to embryo culture medium elicits a significant increase in the survival of transferred embryos to week 12 and live birth. The study results were consistent with a protective effect of GM-CSF on culture-induced embryonic stress. GM-CSF may be particularly efficient in women with previous miscarriage.[31]

Common medium versus advanced in vitro fertilization medium for cryopreserved oocytes in heterologous cycles

GM-CSF plays a crucial role during embryo implantation and subsequent development. Poverini et al. evaluated the effects of embryo cell culture medium, with the inclusion of GM-CSF on embryo development and pregnancy rate. The study results showed that the rate of fertilization and the pregnancy rate were increased using medium added with this cytokine (GM-CSF).[36]

Recommendations

  • For IVF laboratories, the use of devices and equipment, including culture media, is a constant decision-making process, which must be made based on several criteria such as availability, user-friendliness, and clinical results, as well as cost/benefit ratio
  • End-users must be aware of when changes are being made. It is, therefore, essential that culture media manufacturers notify clinics of all changes being made to their products
  • In parallel, the clinics should have a system to validate the products they use in their own laboratory. The results obtained with new media or other methodological changes must be carefully monitored and assessed
  • Results regarding the fertilization rate and embryo quality as well as pregnancy, implantation, and live-born baby rates must be carefully recorded and analyzed. Each IVF unit should develop a system for following up also the health of the offspring and this should be one of the crucial quality control indicators.



   Regulatory Aspects of ART Culture Media Top


  • European Union (EU) would regulate ART culture media as medical devices (Class III) (Medical Device Directive 93/42/EEC, 1993; MEDDEV2.2/4 January 2012)
  • Conformité Européenne marking is a regulatory requirement to show a product is safe, effective, fit for purpose and meets EU regulations
  • If the medium contains a medicinal product or human-derived blood product, such as HSA or antibiotics, the Notified Body also must undertake consultation with a Medicines Competent Authority and/or European Medicines Agency.



   Vitrification Top


Background

Vitrification is now the dominant approach for cryopreservation of human oocytes and embryos. The application of vitrification considerably improved the in vitro and in vivo development of cryopreserved blastocysts and oocytes and opened new perspectives for extended embryo culture, single blastocyst transfer, blastocyst biopsy, and alternative ways for fertility preservation or oocyte donation, respectively.

Cryopreservation protocols have improved the efficiency of ART outcomes in the last few years. However, it remains important to always seek amelioration on cryopreservation protocols and devices to ensure a significant benefit and patients' safety during procedures. We need to find a method that combines a high cooling and warming rate, high survival, and function of cells and tissues and is made in a way that ensures patient safety.[37]

Supporting evidence

Cryodevices

  • These are the devices used to store biological cells at low temperature
  • In ART, they are used for cryopreserving sperm, testicular tissue, oocyte, embryo, blastocyst, and ovarian cortex.


Types

For most embryologists, the two categories are easily distinguishable. Open systems allow, and closed systems eliminate direct contact between the sample-containing medium and liquid nitrogen. Consequently, from a disease transmission point of view, open systems are hypothetically unsafe, and closed systems are safe. There is also a semiclosed type in practice.

Open system[38]

  • Open devices are devices that allow direct contact of the biological sample to be frozen with the cooling solution
  • Generally, using open devices, the achieved cooling rates are approximately 20,000°C–24,000°C/min, which favor good vitrification of the sample
  • The problem is that being in direct contact with the cooling solution there is a risk of pathogen transmission to the biological sample at the time of freezing and a high-risk of cross-contamination in the common cooling containers.


Closed system[34]

  • In such systems, the biological sample is not in connection with the cooling solution at the time of freezing or at the time of storage in the conventional containers
  • This prevents contamination by contact and cross-contamination from shared containers
  • The main feature is that the cooling rate is much lower with these closed devices
  • The closed devices can be locked or sealed in many ways, but most importantly a hermetic seal must be made, preventing entry to the inside and leakage of pathogens to the outside
  • Thermo seal, radiofrequency seal, and ultrasound seal are some of the most used systems that ensure that the sample remains free of contamination.


Factors affecting the vitrification process

The principle of vitrification in cryobiology is to eliminate ice formation totally in the medium that contains the sample, in all phases (cooling, storage, and warming) of the procedure. It can be achieved either by increased cooling and warming rates, or increasing concentration of cryoprotectants; in practical situations, both approaches are applied. The higher the cryoprotectant concentration, the lower the cooling rate required and vice versa. As highly concentrated cryoprotectants may cause toxic and osmotic injury, the preferred strategy is to use the highest possible cooling and warming rates, then to apply the lowest concentration of cryoprotectants that ensures safe ice-free solidification under these circumstances.

Cooling and warming rate

The easiest way to achieve high cooling and warming rates is to use the smallest solution volume and the highest temperature conductivity between the sample-containing medium and the cooling or warming agent, preferably liquid nitrogen for the former purpose.

The requirements and relationships for conditions to achieve satisfactory vitrification in the area of mammalian ART are well displayed in the equation of Yavin and Arav.



Slush nitrogen

  • As a cooling agent, this technique uses slush nitrogen, much colder than LN2 (−196°C vs. −210°C) and with the property of avoiding the Leidenfrost effect. When something is submerged in LN2, bubbles rise to the surface through the device, varying the thermal conductivity from the outside into the inside of the device. This does not happen with slush nitrogen. Slush nitrogen is achieved with a vacuum pump in 5–10 min, and it remains slush for a further 5–10 min before returning to liquid. It was shown for oocytes and embryos that increasing the cooling rate could improve survival rates by up to 37%.


Cross-contamination

In reproductive biology, including mammalian and human-assisted reproduction, no disease transmission caused by liquid nitrogen-mediated cross-contamination, or another cryopreservation-related source, has been reported.



  • No single infection after ART with processed reproductive samples has been reported to date
  • Suitability of closed systems for vitrification is scarce compared with the vast amount of published and unpublished excellent results with open systems.


Automated vitrification of embryos

In IVF clinics, the vitrification is conducted manually by highly skilled embryologists. Processing one oocyte/embryo takes the embryologist 15–20 min, depending on the protocols chosen. Due to poor reproducibility and inconsistency across operators, the success and survival rates also vary significantly.[39]

The process of realizing robotic vitrification (RoboVitri) is ongoing and ultimately aims to standardize clinical vitrification from manual operation to fully automated robotic operation.[35]

Tests on mouse embryos demonstrated that the system could perform vitrification with a throughput at least three times that of manual process and a high survival (88.9%) and development rate (93.8%).[35]

Novel automatic vitrification devices are capable of producing high survival rates of oocytes and embryos. Researchers anticipate that as the demand for vitrification of gametes, embryos, and reproductive tissues increases worldwide, the availability of an automated vitrification device will become indispensable for standardization, simplification, and reproducibility of the entire process.[40]
Table 6: Embryo vitrification experimental results

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Consensus

  • Open devices can be used to vitrify embryos as no incidence of cross-contamination is reported so far
  • The aim should be to use a system with no chances of cross-contamination
  • Closed devices are hermetic, can be used, but need more data to support the use in the laboratory
  • Separate storage containers for infective samples should be used
  • Automation is experimental but could be the future for vitrification.



   Assisted Hatching Top


Background

Assisted hatching (AH) involves the artificial thinning or breaching of the zona pellucida (ZP) and has been proposed as one technique to improve implantation and pregnancy rates following IVF. Several methods have been employed over the past few years to improve implantation rates in patients undergoing IVF/ICSI. Hatching of the zona was performed mechanically or chemically or by using a laser. All these methods aim to create a hole in the ZP or to thin a part of it, for assisting the embryo in hatching out at the time when the blastocyst expands, and it is ready to implant.[41]

Supporting evidence

Few studies in the literature compared laser with other techniques of AH. In 2002, Hsieh et al. compared 85 women aged ≥38 years, whose embryos had laser-AH, with 56 women of the same age group whose embryos had chemical AH. They concluded that laser hatching is more effective than the chemical method in enhancing the implantation and pregnancy rates of women with advanced age. The implantation rate of transferred embryos was significantly improved in the laser group, whereas pregnancy rates showed a tendency to increase, even though the difference did not reach statistical significance.[41]

Recommendation on assisted hatching-LASER




   Time-Lapse Imaging Top


Background

Time-lapse is defined as an intervention that comprises undisturbed embryo culture and the prospective use of visual information obtained by time-lapse monitoring (TLM) for embryo evaluation. Time-lapse recording introduces several dynamic morphological parameters for embryo evaluation. However, the data analysis and comparison between studies are complicated due to diverging nomenclature and definitions of events and their timing. Consensus on how to collect and report data is therefore desirable.[42]
Table 7: Potential advantages and disadvantages of the time-lapse imaging method

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Supporting evidence

Noninferiority as compared to standard embryo assessment

Application of TLM together with embryo-evaluating algorithm was associated with a significantly higher ongoing pregnancy rate, a significantly lower early pregnancy loss and a significantly higher live birth rate. Extremely close agreement (ICC = 0.99) was found for dynamic parameters including the timing of the following: pronuclei breakdown, completion of blastocyst hatching and the appearance and disappearance of the first nucleus after the first division. Observations of cleavage divisions were strongly correlated (ICC >0.8), indicating close agreement. Measurements of the static morphologic parameters, i.e., multi-nucleation and evenness of blastomeres at the 2-cell stage showed fair-to-moderate understanding (ICC ≤0.5).

Review of the literature on morphokinetics shows that the range of timing of events in embryo development may provide additional clues to embryonic potential without the need for unnecessary exposure. There are several retrospective studies evaluating specific cell cycle kinetic parameters evaluated with time-lapse imaging associated with blastocyst formation and/or pregnancy outcomes. Research has suggested that embryos cultured in TLM systems have a higher implantation potential. Recently, a study has used time-lapse imaging to assess embryo quality and evaluation of known implantation data scores and concluded that quality assessment of the embryos was better with TLM than with the conventional method.[44]

Proposed morphokinetic variables

Definitions for the dynamic monitoring of human preimplantation embryo development are given in [Figure 1].[45]
Figure 1: Definitions for the dynamic monitoring of human preimplantation embryo development

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Ploidy concordance

Campbell et al. showed that embryos with multiple aneuploidies showed delay at the initiation of compaction (tSC; median 85.1 h postinsemination [hpi]; P = 0.02) and the time to reach full blastocyst stage (tB; median 110.9 hpi, P = 0.01) compared with euploid embryos (tSC median 79.7 hpi, tB median 105.9 hpi).[46] Embryos having single or multiple aneuploidies (median 103.4 hpi, P = 0.004 and 101.9 hpi, P = 0.006, respectively) had delayed initiation of blastulation compared with euploid embryos (median 95.1 hpi). Morphokinetic parameters should not be used yet as a surrogate for PGT to determine embryo ploidy in vitro [Table 5].

Currently, there are numerous examples where adjunct treatments are used in the absence of evidence-based medicine and often at an additional fee. Robust studies are needed to confirm the safety and efficacy of any adjunct treatment or test before they are offered routinely to IVF patients.

Consensus on indications and outcomes



Consensus on the benefits of time-lapse imaging

  • It improves the flexibility of assessment
  • It enables documentation and traceability of the laboratory's handling and selection of embryos
  • Training of laboratory personnel is recommended
  • Validation of the laboratory's embryo assessment is recommended.



   Spindle View Top


Background

The meiotic spindle (MS) of human oocytes in metaphase II (MII) is a temporary dynamic structure consisting of microtubules that are associated with the oocyte cortex and its network of subcortical microfilaments. The MS microtubules are linked to the kinetochores of the chromosomes and participate in segregation during meiosis. It is known that the oocyte MS integrity is essential for chromosome segregation and that it is susceptible to various factors such as ageing, thermal changes, insufficient oxygen supply during culture, and oocyte manipulation. The MS plays a vital role in the oocyte during chromosome alignment and separation at meiosis.




   Consensus for indications Top





   Training for Embryologist Top


Why is training for add-ons necessary?

  • IThere is growing evidence to suggest that add-ons are being offered to patients, without conclusive evidence to date
  • ISpecific add-on treatments are readily available and can be provided in any laboratory scenario
  • ITo maintain the transparency about the add-on treatments, they offer, including the potential costs, to ensure their chance of having a baby


  • ITo ensure that the validation of the laboratory and staff.


The recommendation for training and process validation: An expert panel survey analysis

The expert panel survey was conducted on 151 respondents; 74.3% of the survey participants recommended imparting the training to embryologists before using the following methods or procedures [Figure 2].
Figure 2: Survey responders in support of training and process validation

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Sperm DNA fragmentation

  • IAbout 91.4% of respondents believed that embryologists should be trained for sperm DNA fragmentation test
  • About 67.7% (100 responded out of 150) respondents believed hands-on training would be beneficial followed by on the job training (33.3%, 50 responded out of 150), workshop/observer course (22.7%, 34 responded out of 150), and then outsource dependency [Figure 3].
Figure 3: Survey analysis of the recommended training types

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Whether training is recommended for sperm selection modalities?

Q.1. Which of the sperm selection modalities requires training?

Based on the survey 79.9 % felt IMSI required training before use, 64.4% felt PICSI required training, 55 and 54.4 % felt microfluidics and. MACS required additional training
Figure 4: Survey responders in support of training for sperm selection modalities

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Q.2a. Which type of training is recommended for sperm selection modalities?

IMSI: Hands-on (61.6%), on the job (23.2%) and workshop/observership (15.2%) training.

Q.2b. Which type of training is recommended for PICSI?

Hands-on course (55.3%), on the job (27.3%), and workshop/observership (17.3%).

Q.2c. Which type of training is recommended for microfluidics/MACS?

Hands-on course (39.2%), workshop/observership (31.1%), and on the job (29.7%)
Figure 5: Survey responders in support of training for sperm selection modalities

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Whether training is recommended for sperm selection and preparation for surgically retrieved sperm?

Q.3 Which of the following procedures requires training for sperm selection and preparation?

TESE (86.4%), TESA (75.5%), and PESA (49.7%).
Figure 6:

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Q.4 What type of training is recommended?

Hands-on job (75%), hands-on course (50.7%) and workshop/observership (29.1%).

Q.5a. Do you think a theoretical understanding should be imparted to embryologists before they use an advanced culture media?

Calcium ionophore (73.5%), embryo glue (51.7%), theophylline (51%), and embryogen (47.6%).
Figure 7: Survey responders in support of training to embryologists before they use an advanced culture media

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Q.6a. Do you think specialized hands-on training should be conducted before opting for oocyte vitrification?

Yes (85.4%), maybe (9.9%), and no (negligible response).

Q.6b. Do you think specialized hands-on training should be conducted before opting for blastocyst vitrification?

Yes (77.7%), maybe (14.9%), and no (negligible response).
Figure 8: Survey responders in favor of training to embryologists for oocyte and blastocyst vitrification

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Assisted hatching

Q.7. Do you think specialized training should be conducted before opting for AH?

Yes (74.7%), maybe (14.7%), and no (10.7%).
Figure 9: Survey responders in favor of training to embryologists for assisted hatching

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Q.8 What type of training do you recommend for AH?

Hands-on (66.7%), on the job (33.3%), and workshop/observership (22.7%).
Figure 10: Survey responders in favor of the type of training for assisted hatching

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Time-lapse and electronic witnessing

Q.9. Do you think specialized training should be conducted before using time-lapse and electronic witnessing?

Yes, 75.5%, maybe 21.2% and no negligible response.
Figure 11: Survey responders in favor of imparting training before using time-lapse and electronic witnessing systems

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Q.10. Do you see any harm in utilizing these procedures without training? (multiple choices)

Yes, identification error 30.1%, poor embryo selection 26.7%, more exposure on embryo 24.7%, and unable to utilize the technology 87.7%.
Figure 12: Negative consequences of lack of training

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Q.11 Do you think specialized training should be conducted before using spindle view?

Yes, 74.3%, maybe 21.6%, and no (negligible response).
Figure 13: Survey responders in favor of imparting training before using spindle view

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Q.12. What are the benefits of additional training and validation? (multiple choices)

Utilize technology to fullest 63.1%, better identification of intended patients 36.2%, better outcomes with the selected technology 73.2%, more confidence in using technology 63.8%, and no benefit 1.3%.
Figure 14: Overall benefits of the training and process validation

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Consensus





 
   References Top

1.
Treatment Add-Ons. Available from: https://www.hfea.gov.uk/treatments/explore-all-treatments/treatment-add-ons/. [Last accessed on 2019 Nov 01].  Back to cited text no. 1
    
2.
Nwaru BI, McCleary N, Erkkola M, Kaila M, Virtanen SM, Sheikh A. Assisted reproductive technology and risk of asthma and allergy in the offspring: Protocol for a systematic review and meta-analysis. BMJ Open 2016;6:e010697.  Back to cited text no. 2
    
3.
Kleijkers SH, Mantikou E, Slappendel E, Consten D, van Echten-Arends J, Wetzels AM, et al. Influence of embryo culture medium (G5 and HTF) on pregnancy and perinatal outcome after IVF: A multicenter RCT. Hum Reprod 2016;31:2219-30.  Back to cited text no. 3
    
4.
Zandstra H, Brentjens LB, Spauwen B, Touwslager RN, Bons JA, Mulder AL, et al. Association of culture medium with growth, weight and cardiovascular development of IVF children at the age of 9 years. Hum Reprod 2018;33:1645-56.  Back to cited text no. 4
    
5.
Lacamara C, Ortega C, Villa S, Pommer R, Schwarze JE. Are children born from singleton pregnancies conceived by ICSI at increased risk for congenital malformations when compared to children conceived naturally? A systematic review and meta-analysis. JBRA Assist Reprod 2017;21:251-9.  Back to cited text no. 5
    
6.
Palermo GD, Neri QV, Takeuchi T, Squires J, Moy F, Rosenwaks Z. Genetic and epigenetic characteristics of ICSI children. Reprod Biomed Online 2008;17:820-33.  Back to cited text no. 6
    
7.
Källén B, Orvar F, Karl-Gösta N, et al. Asthma in Swedish children conceived by in vitro fertilization. Arch Dis Child 2012;98:89-90.  Back to cited text no. 7
    
8.
Bonduelle M, Van Assche E, Joris H, Keymolen K, Devroey P, Van Steirteghem A, et al. Prenatal testing in ICSI pregnancies: Incidence of chromosomal anomalies in 1586 karyotypes and relation to sperm parameters. Hum Reprod 2002;17:2600-14.  Back to cited text no. 8
    
9.
Berntsen S, Söderström-Anttila V, Wennerholm UB, Laivuori H, Loft A, Oldereid NB, et al. The health of children conceived by ART: 'The chicken or the egg? Hum Reprod Update 2019;25:137-58,  Back to cited text no. 9
    
10.
Deenadayal Mettler A, Govindarajan M, Srinivas S, Mithraprabhu S, Evenson D, Mahendran T. Male age is associated with sperm DNA/chromatin integrity. Aging Male 2019;9:1-8.  Back to cited text no. 10
    
11.
Zini A, Sigman M. Are tests of sperm DNA damage clinically useful? Pros and cons. J Androl 2009;30:219-29.  Back to cited text no. 11
    
12.
Meseguer M, Santiso R, Garrido N, García-Herrero S, Remohí J, Fernandez JL. Effect of sperm DNA fragmentation on pregnancy outcome depends on oocyte quality. Fertil Steril 2011;95:124-8.  Back to cited text no. 12
    
13.
Bach PV, Schlegel PN. Sperm DNA damage and its role in IVF and ICSI. Basic Clin Androl 2016;26:15.  Back to cited text no. 13
    
14.
Pratap H, Hottigoudar SY, Nichanahalli KS, Chand P. Assessment of sperm deoxyribose nucleic acid fragmentation using sperm chromatin dispersion assay. J Pharmacol Pharmacother 2017;8:45-9.  Back to cited text no. 14
[PUBMED]  [Full text]  
15.
Collins AR, Oscoz AA, Brunborg G, Gaivão I, Giovannelli L, Kruszewski M, et al. The comet assay: Topical issues. Mutagenesis 2008;23:143-51.  Back to cited text no. 15
    
16.
Al Omrani B, Al Eisa N, Javed M, Al Ghedan M, Al Matrafi H, Al Sufyan H. Associations of sperm DNA fragmentation with lifestyle factors and semen parameters of Saudi men and its impact on ICSI outcome. Reprod Biol Endocrinol 2018;16:49.  Back to cited text no. 16
    
17.
Vaughan DA, Sakkas D. Sperm selection methods in the 21st century. Biol Reprod 2019;101:1076-82.  Back to cited text no. 17
    
18.
Sakkas D, Ramalingam M, Garrido N, Barratt CL. Sperm selection in natural conception: What can we learn from Mother Nature to improve assisted reproduction outcomes? Hum Reprod Update 2015;21:711-26.  Back to cited text no. 18
    
19.
Surgical Sperm Retrieval. Available from: https://www.washington.edu/urology/surgical-sperm-retrieval/. [Last accessed on 2019 Sep 01].  Back to cited text no. 19
    
20.
Esteves SC, Miyaoka R, Agarwal A. Sperm retrieval techniques for assisted reproduction. Int Braz J Urol 2011;37:570-83.  Back to cited text no. 20
    
21.
Grynberg M, El Hachem H, de Bantel A, Benard J, le Parco S, Fanchin R. In vitro maturation of oocytes: Uncommon indications. Fertil Steril 2013;99:1182-8.  Back to cited text no. 21
    
22.
Practice Committees of the American Society for Reproductive Medicine and the Society for Assisted Reproductive Technology. In vitro maturation: A committee opinion. Fertil Steril 2013;99:663-6.  Back to cited text no. 22
    
23.
Vanden MF, Nikiforaki D, De Gheselle S, et al. Assisted oocyte activation is not beneficial for all patients with a suspected oocyte-related activation deficiency. Hum Reprod 2012;27:1977-84.  Back to cited text no. 23
    
24.
Sunde A, Brison D, Dumoulin J, Harper J, Lundin K, Magli MC, et al. Time to take human embryo culture seriously. Hum Reprod 2016;31:2174-82.  Back to cited text no. 24
    
25.
Gardner DK. Changes in requirements and utilization of nutrients during mammalian preimplantation embryo development and their significance in embryo culture. Theriogenology 1998;49:83-102.  Back to cited text no. 25
    
26.
Kleijkers SH, van Montfoort AP, Bekers O, Coonen E, Derhaag JG, Evers JL, et al. Ammonium accumulation in commercially available embryo culture media and protein supplements during storage at 2-8°C and during incubation at 37°C. Hum Reprod 2016;31:1192-9.  Back to cited text no. 26
    
27.
Quinn P. The development and impact of culture media for assisted reproductive technologies. Fertil Steril 2004;81:27-9.  Back to cited text no. 27
    
28.
Ménézo Y, Lichtblau I, Elder K. New insights into human pre-implantation metabolism in vivo and in vitro. J Assist Reprod Genet 2013;30:293-303.  Back to cited text no. 28
    
29.
Antibiotics in Cell Culture: Friends or Enemy? Available from: https://www.labclinics.com/en/antibiotics-cell-culture-friends-enemy/. [Last accessed on 2020 Jan 01].  Back to cited text no. 29
    
30.
Zhu J, Li M, Chen L, Liu P, Qiao J. The protein source in embryo culture media influences birthweight: A comparative study between G1 v5 and G1-PLUS v5. Hum Reprod 2014;29:1387-92.  Back to cited text no. 30
    
31.
Ziebe S, Loft A, Povlsen BB, Erb K, Agerholm I, Aasted M, et al. A randomized clinical trial to evaluate the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) in embryo culture medium for in vitro fertilization. Fertil Steril 2013;99:1600-9.  Back to cited text no. 31
    
32.
Renzini M, Canto M, Coticchio G, et al. Clinical efficiency and perinatal outcome of ART cycles following embryo culture in the presence of GM-CSF in patients with miscarriage or early pregnancy loss history. Hum Reprod 2013;28:i160-202.  Back to cited text no. 32
    
33.
Sfontouris IA, Lainas GT, Sakkas D, Zorzovilis IZ, Petsas GK, Lainas TG. Non-invasive metabolomic analysis using a commercial NIR instrument for embryo selection. J Hum Reprod Sci 2013;6:133-9.  Back to cited text no. 33
[PUBMED]  [Full text]  
34.
Siristatidis C, Vogiatzi P, Salamalekis G, Creatsa M, Vrachnis N, Glujovsky D, et al. Granulocyte macrophage colony stimulating factor supplementation in culture media for subfertile women undergoing assisted reproduction technologies: A systematic review. Int J Endocrinol 2013;2013:704967.  Back to cited text no. 34
    
35.
Singh N, Gupta M, Kriplani A, Vanamail P. Role of Embryo Glue as a transfer medium in the outcome of fresh non-donor in-vitro fertilization cycles. J Hum Reprod Sci 2015;8:214-7.  Back to cited text no. 35
[PUBMED]  [Full text]  
36.
Poverini R, Lisi R, Lisi F, Berlinghieri V, Bielli W, Carfagna P, et al. Common medium versus advanced IVF medium for cryopreserved oocytes in heterologous cycles. Cell Death Discov 2018;4:34.  Back to cited text no. 36
    
37.
The Problem of Contamination: Open vs. Closed vs. Semi-Closed Vitrification Systems. Available from: https://www.intechopen.com/books/current-frontiers-in-cryopreservation/the-problem-of-contamination-open-vs-close-vs-semi-close-vitrification-systems. [Last accessed on 2020 Jan 01].  Back to cited text no. 37
    
38.
Vajta G, Rienzi L, Ubaldi FM. Open versus closed systems for vitrification of human oocytes and embryos. Reprod Biomed Online 2015;30:325-33.  Back to cited text no. 38
    
39.
Liu J, Shi C, Wen J, et al. Automated Vitrification of Embryos. https://ieeexplore.ieee.org/xpl/aboutJournal.jsp?punumber=100: IEEE Robotics & Automation Magazine https://www.researchgate.net/journal/IEEE-Robotics-Automation-Magazine-1558-223X. [Last accessed on 2020 Jan 01].  Back to cited text no. 39
    
40.
Arav A, Natan Y, Kalo D, Komsky-Elbaz A, Roth Z, Levi-Setti PE, et al. A new, simple, automatic vitrification device: Preliminary results with murine and bovine oocytes and embryos. J Assist Reprod Genet 2018;35:1161-8.  Back to cited text no. 40
    
41.
Makrakis E, Angeli I, Agapitou K, Pappas K, Dafereras A, Pantos K. Laser versus mechanical assisted hatching: A prospective study of clinical outcomes. Fertil Steril 2006;86:1596-600.  Back to cited text no. 41
    
42.
Kirkegaard K, Agerholm IE, Ingerslev HJ. Time-lapse monitoring as a tool for clinical embryo assessment. Hum Reprod 2012;27:1277-85.  Back to cited text no. 42
    
43.
Insua MF, Cobo AC, Larreategui Z, Ferrando M, Serra V, Meseguer M. Obstetric and perinatal outcomes of pregnancies conceived with embryos cultured in a time-lapse monitoring system. Fertil Steril 2017;108:498-504.  Back to cited text no. 43
    
44.
Seshadri S, Saab W, Serhal P. Time lapse imaging of embryos is useful in in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI) treatment: FOR: Time-lapse monitoring of embryos. BJOG 2019;126:287.  Back to cited text no. 44
    
45.
Ciray HN, Campbell A, Agerholm IE, Aguilar J, Chamayou S, Esbert M, et al. Proposed guidelines on the nomenclature and annotation of dynamic human embryo monitoring by a time-lapse user group. Hum Reprod 2014;29:2650-60.  Back to cited text no. 45
    
46.
Campbell A, Fishel S, Bowman N, Duffy S, Sedler M, Hickman CF. Modelling a risk classification of aneuploidy in human embryos using non-invasive morphokinetics. Reprod Biomed Online 2013;26:477-85.  Back to cited text no. 46
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]



 

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    Abstract
    Introduction to ...
    The Health of th...
    DNA Fragmentatio...
    Sperm Selection ...
    Surgical Sperm R...
    Culture Media Ad...
    Regulatory Aspec...
   Vitrification
   Assisted Hatching
   Time-Lapse Imaging
   Spindle View
    Consensus for in...
    Training for Emb...
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