CNV and relation with unexplained miscarriage

Lack of knowledge on etiology of spontaneous miscarriage .Doctors ignorance on a given case of miscarriage: Miscarriage in cases of much wanted pregancy is a curse to the couple and family members as well. Unfortunately, as members are aware that in many cases the cause of spont abortion remains unclear. The concerned physician is sometimes accused; This blame on doctor is mote in cases of abortion following ART procedures where hopes were sky high. But question is have we improved our knowledge on etilogy in past three decades so that an acceptable remedy or at least an explanation become acceptable to couple? Do we have any new informations on miscarriage may be we  don’t  have remedy or drugs to control that abnormality leading to abortion.

Our hands are tied: We just can’t help the couple: From last five decades we knew that embryonic numerical and structural chromosomal abnormalities are the most common cause of early pregnancy loss. Embryonic or parental 1) chromosomal abnormalities,2)  infectious disorders 3) autoimmune diseases 4)  sperm quality, 5) antiphospholipid syndrome, 6) uterine anomalies,7)  thrombophilias,8)  endocrine disorders, 9)  lifestyle issues and above all 10)  endometritis 11) endometriosis  are all considered potential causes of miscarriage. Early pregnancy loss, or miscarriage, is the most frequent complication in first‐trimester pregnancy, occurring in 10–15% of all clinically recognized pregnancies. But what worries the practicing obstetrician is that  embryonic numerical and structural chromosomal abnormalities are the most common cause, accounting for more than 50% of miscarriages.  Our hands are tied in such cases except being depressed for couple of hours.

Ray of hope!!!! But what do we mean by “Copy number Variations”? What is role in spont early abortions??Ans  :CNV = Copy-number variations ,.The role / prevalence in causation of  submicroscopic copy‐number variations (CNVs) in early pregnancy loss is still unclear, and little is known about the critical regions and candidate genes for miscarriage, because of the large size of structural chromosomal abnormalities. However the potential miscarriage‐associated submicroscopic CNVs and critical regions of large CNVs as well as candidate genes for miscarriage have been assessed in last two decades by many researchers across te globe,. Clinically significant chromosomal abnormalities were identified in 59.1%

How many members believe that   QF- PCR technology applied in POC will help the couple in future pregancy?? Do we have drugs to prevent such “COPY NUMBER VARIATIONS”??   

QF- PCR technology is in vogue for last 3 decades and such a technology has an important role to study CNVs in the  fresh miscarriage specimens .by using quantitative fluorescent polymerase chain reaction/CNV sequencing or chromosomal microarray analysis. It is known that embryonic major chromosomal abnormalities are the most common cause of miscarriage. Our results demonstrate the role of copy‐number variations (CNVs) in the etiology of miscarriage.

 IN search of a geneticist who can highlight our queries on Abnornal genes or variety of mutation disorders, The coupe is prepared to pay more so if such an even can be corrected by a kind hearted geneticist with reasonable accuracy. What do we mean by “next‐generation sequencing (NGS)” ?? Ans;-, With the wide application of high‐resolution molecular techniques, including chromosomal microarray analysis (CMA) and next‐generation sequencing (NGS), submicroscopic copy‐number variations (CNVs) have been recognized to be associated with a wide range of human diseases, including congenital anomalies and neurodevelopmental disorders

In recent years, submicroscopic CNVs have also been observed in cases of miscarriage.  Besides, although large CNVs are known to cause miscarriage, there are few reports of the specific regions critical for miscarriage and miscarriage candidate genes in CNV regions.

The incidence and distribution of chromosomal abnormalities detected by CMA and the NGS‐based method, CNV sequencing (CNV‐seq), The potential miscarriage‐associated submicroscopic CNVs and critical regions of large CNVs is possible.. It is now possible to identify potential miscarriage candidate genes from critical regions of miscarriage‐associated CNVs using gene‐prioritization analysis

How best to select sperm which are of good DNA content and has good morphology i,e potentiality to fertilize?

Newer   modalities    for sperm  selection and how  we can implement    them in the laboratory .

The physiological barrier or the natural selection that the sperm   has to undergo  in order to fertilize the oocyte  What are the physiological barrier or the natural selection that the sperm   has to undergo  in order to fertilize the oocyte?? Ans:- In the year     1992  it was showed by the researchers that  there was a   dramatic reduction   in the  number of sperms   recovered from the fallopian  tube  when artificially    inseminated.

 This    reduction clearly brought about a change in the thought process at that time and researchers where now focusing on the physiological barrier or the natural selection that the sperm   has to undergo  in order to fertilize the oocyte    . These   physiological  barriers   include the acidic    ph  of the vagina , cervical    mucus   response to changes   in the uterine environment   going from   the  uterus to the tubes the binding of the sperms to the zona pellucida  ect.

These mechanisms selectively reduce the amount of sperms  reaching   the egg and fertilizing   it but  amount   of sperms   reaching the egg   and fertilizing it but the question  now is   how to make   the sperm  selection equivalent to  the natural and how to improve   the existing   modalities  so as to select   the single   best sperm   the millions  that are   ejaculated.

Even   though IVF    in now    four decades   old we   still  don’t   understand  the how   effective   these sperms really   are and most judgments are basically  based  on microscopic  evaluation which   is not  the best way to judge the potential of the sperm also we know for a fact    that all motile    sperms      don’t have  the potential  to fertilise the   oocytes   and judging    the sperms   based on   motility alone again  wouldn’t   be the best way to select.

Now logically  if we try to mimic the natural    selection in the ivf   laboratory our   sperm selection     would definitely improve and hopefully     would give us better outcomes  . In this     chapter   we will try and cover    the newer   modalities    for sperm selection and how  we can implement    them in the laboratory .

 What is peculiar or specialty in Sperms  cells in any animals?? Ans:- Sperm cells (male gametes) are  by  far be considered  as the most  specialised cells in   the body they are  programmed to do the most important    task   which is to create life.  Different      parts of the sperm have different functions during    the process of fertilisation  . The 1) head  carries   the DNA   content   which has to    be delivered   to the egg the 2) mid   piece   stores   the energy  creating mitochondria    3) the  tail   carries   out the flagellar   propulsion ,

AS such the  grading and selecting    based on these  three  important components   is something  which is generally  followed worldwide.

   In ICSI procedure we know that a heavy responsibility lies on embryologist to select the best sperm for injection in the oolemma. How this selection is done? Is such sperm are the best sperms?? Now we should remember that till now most ART center  sect sperm for injection based on sperm motility   or  the sperm morphology . Such sperms are are usually selected as full marks for injection to oocytes. But is that all??  Then why there are so many cases of “Failure to progress normally after blastocyst stage” :- after implantations in ICSI procures??   The procedure of ICSI –The outcome depends mainly on judgement    of the embryologists : Having admitted that may one ask what are the limitations of sperm selection in ICSI as of now?? Ans: The limitations are even today most of the ART ( ICSI-  IVF ) the andrologist /embryologist has the option to select the best sperm under. But the limitations is that of all available methods to select the best sperm in ICSI procedure of all the  available  techniques    basically   depend on    selection the sperm   based on motility      or on the  morphology     but these don’t   really mimic any   of the natural   processes. The sperms can  then be chosen by the embryologist   on the ICSI  manipulator thus adding to the selection  process. But again these   modalities are only based  on the judgement    of the embryologist which isn’t    standardized and varies   heavily between users. The newer techniques    which have come up    and have started to  show a positive  impact as fear as results   are concerned are PICSI   or hyaluronan   binding    capacity   .

 IMSI  intra cytoplasmic   morphologically selected sperm    injection,  Microfluidies,  Magnetic   activated  sperm     sorting  and Omics.

Sperm plasma   membrane remodeling:-  PICSI:-  The  sperm plasma   membrane remodeling   is an important    step  in preparing the sperm to fertilise the egg during this processes specific    receptors  form like the zone pellucida   receptors   and the hyaluronic   acid receptors  which aid in   cumulus binding . The principle  behind   the use of PICSI is that when a mature   sperm reaches the Hyamuronic acid rich  cumulus complex surrounding the human     egg such sperm    binds   and  initiates  the final fertilisation . We should remember that the only   fully matured     sperm have developed HA  receptors  and can bind to the zone pellucida .  Immature sperm   cannot  bind to HA    .

What about sperm DNA fragmentation ?? Researchers have also noticed that failure to fertilize injected sperms in ICSI / Recurrent early pregnancy loss  may follow  in women whose husband’s sperms exhibit  have higher rates of DNA   damage and a  higher occurrence of carrying  abnormal numbers  of chromosomes . Thus  patients that  have   a high DNA  fragmentation  index or have    suffered   from recurrent miscarriages  or have poor   quality  of embryos  in multiple   cycles   can benefit   from this  technique    carried    out a Cochrane   review  in 2014  which came  to the conclusion  that more evidence   is required to come  to a conclusion  to whether or not  PICSI is actually beneficial or not .

IMSI

How best to avoid poor sperms(morphologically sperms) IMSI: Injection of morphologically selected sperms: Humans   ejaculate a considerably large number of morphologically abnormal sperms . We have a strict  criteria    to grade  the morphology    of the sperm  and also   to decide  on the treatment    options for the patients . Most  of the times  patients with  poor morphology are directed   towards        an ICSI      cycle justification being that the   embryologist  can choose   the morphologically  normal   sperms    to fertilise  the oocyte  . However recent papers     have shown  that even the sperms    which appear normal   in ICSI   can have     multiple  defects   as shown   .

Gross   evaluation    of sperms   on ICSI   magnification  that is  400x  isn’t   sufficient    to defect   subtle   aberrations which according    to some have   higher chances  of transmitting genetic as well as chromosomal diseases  . Also   there is a need to standardise the scoring system   with IMSI   as of now there   are 4  grades in which the sperms  can be classified  into the according    to the current  grading   more standardized   Cochrane   review  on IMSI  states   that      there was no significant   difference in live birth  between IMSI  and ICSI  also  no significant   difference   in pregnancy   rates or  miscarriages rtes But we   need to study   the prognosis of an IMSI  cycle vs  and ICSI   cycle in    patients who    actually have poor    parameters and  then only  we would be able to justify  the use of IMSI.

   as such

Sperm    vacuoles   have also   been researched and have   mostly been associated  with a higher DNA  fragmentation     index  but this   association  still  needs  to be proven  

.

,Application of MACS   in IVF

Improves   fertilisation potential

Increases cleavage  and pregnancy   rates

Reduces   sperm DNA  fragmentation

Improves live  birth rate

But  again more systematic  reviews are needed to back   these  claims 

MICROFLUIDICS

 Microfluidics  is an exciting  technology    which   now takes   into consideration  the biophysical and biochemical  milieu which    the sperm encounters in the   Female   reproductive  tract Studies  have shown   an increase  in motile   percentage of sperm    from below  60%  to 90%  they have  also  shown   better DNA   integrity post sorting      system could  reduce  the treatment   time for   intracytoplasmic .

Conclusion

Even   though   the technological  advances  are occurring at a rapid   pace the biggest challenge  that still  remains   is justifying   these technologies  and proving   their  efficacy  with  long term    data   to support it .

 Sperm   selection  brings  forth a very   exciting   new frontier in IVF  and it takes  us closer to  achieving   our aim of    having   a single   live birth   from a single  embryo   transfer. It also   presents   us with   various challenges  as far as male  infertility   and its  management  is concerned.

OHSS -Ovarian hyperstimulation syndrome : in IVF cycles: How to prevent & treat ?

ABC of  prediction of  OHSS in assisted reproduction

 Ovarian   hyper   stimulation   syndrome  is one of the main  iatrogenic complications of controlled  ovarian    hyperstimulation in Assisted   reproduction. It occurs  after triggering ovulation   with  exogenous  human   chorionic   gonadotropin   and may be  aggravated  by pregnancy  . The incidence    of moderate OHSS   with assisted    reproductive   techniques is  0.1-3%  OHSS can have   potentially fetal  consequences in  3/100000     stimulated  women.

Some of the factors that increase    the risk  of developing  OHSS are    younger   age low BMI  previous history    of OHSS history of    exaggerated   response  to gonadotropins   in intrauterine  insemination cycles and presence  of polycystic ovarian   syndrome.

Pathophysiology : Administration of hCG   leads  to an increase   in various interleukins causing increased capillary permeability   resulting   in fluid   shift  from  intravascular  to the extra vascular compartment . This leads to ascites   occasionally    pleural effusion   and enlarged    ovaries.  The rapid fluid  shift   also causes  hypovolemia    and hemoconcentration  . The    haemoconcentration   that occurs  is reflected in the rise of  hematocrit level

Severity  of OHSS   is classified according to the criteria suggested by Golan  et al

Mild OHSS : nausea vomiting   ovarian  size < 5 cm

Moderate OHSS: abdominal distension    ascites  along   with  nausea and vomiting    with ovarian   size more than  5 cm  

Severe OHSS : massive    ascites  haemoconcentration    breathlessness Oliguria enlarged   ovaries.

 REVIEW OF LITERATURE

 According to the royal college of obstetricians  and  Gynae cologists   OHSS   complicates     almost 33%  of cycles   of ovarian    stimulation and incidence of severe  form    varies    between   3 and 8 5  of IVF cycles.

OHSS  adds   to the emotional and   financial burden     of IVF and   hence  we felt   a need  to search   for a simple marker    which   could    help predict   development   of OHSS and manage    the situation  better    avoiding   the associated   morbidity

Risk    factors    suggested     for OHSS  are PCOS   age    multifollicular response   high estradiol    on the day of  HCG    and lean   habitus.

A study  on OHSS found  that the haemoconcentration established  by  an increase   of hematocrit of more  than 35%  on the day of OPU   was more likely to result    in OHSS   when the haemocrit  on the day  of OPU  was  more than 40%   almost 60%  of the subjects    developed OHSS. When     plotted on a receiver   operator  characteristic  curve  the   haematocrit on   the   day of OPU had a 77.8% sensitivity    for predicting OHSS  but a poor   specificity  .

Sensitivity    of the haematocrit level   on the day of ET  in predicting OHSS  was 85.2%   with the diagnostic accuracy  of   83.5%  Due to the low prevalence of OHSS the positive    predictive  value was 52.3%   however  when   th haematocrit  on the day  of ET   was less  than 35%   the chances  of developing     OHSS  are small with a negative  predictive   value  of 81.8%   Predicting     the occurrence of OHSS therefore justifies the use  of hematocrit  as a simple test.

Another   predictor  of OHSS suggested   by Verit et al  is the neutrophils lymphocyte ratio which  was found superior   to platelet  lymphocyte ratio with    a sensitivity  of 85%    and specificity     of 785  However it can be argued  that haemotocrit is a   much simpler and cheaper  parameter   to analyse compared    to the above   ratios.

Quantitative 3D  Doppler angiography has also   been studied as a predictor of OHSS There was no demonstrable  increased  ovarian  blood flow between  the women   who developed  OHSS  and those   who did not     thus     disproving   the hypothesis.

AMH is a more recent  kid on the block Apart   from being  considered  a good  predictor   of the ovarian   response   to controlled   ovarian   hyperstimulation  its  role in predicting OHSS  is also encouraging . In the study  by Lee   et al AMH   was found to be    a better  predictor of OHSS  than age  and NMI  as well as   marginally  better   than the estradiol   levels  on the day of HCG.

Haematocrit is a simple   inexpensive and  fairly  accurate  test for predicting the development of OHSS  in ART. There   should   be a thorough  assessment   and a low threshold to  defer embryo  transfers in women    with a hematocrit of more   than 35%    on the day  of ET  or those   showing   an increase  

Cabergolin in the treatment of Pelvic/ extra pelvic endometriosis

Members practice experience on cabergolin tr for endometriosis:: Role of cabergoline in the treatment of endometriosis..when, how much, how long...Any acqud Cardiac diseases induced by cabergolin??

cabergoline inhibits endometrial VGEF,. So, it reduces endometrial activity located anywhere in the body anywhere .

the question is good. Cheap, easily available and as The action mode is down-regulation of VEGF/VEGFR-2, pro-angiogenic cytokines and plasminogen activator inhibitor-1 within the lesions. There has been very few human studies, except from Gomez et al., in humans and long term use is known to cause cardiac valvular regurgitation. Most studies are on mice models and extrapolating the data, as to the optimal dose, duration and end-point is not known. AS such cabergolin should , till further data pours in should not be, according me the drug of first choice as t has to be ingested for months together, I am unwire if such cargoes problems does occur in micro or microadenoma white cabergolin is used for moths together.

For a distressed woman unhappy with dienogest due to its known 5 side effects & contraindications of dienogest treatment like cabergolin can be offered.

Like

Unconventional treatment of pelvic endometriosis s

Unconventional treatment of endometriosis: Poor woman’s treatment !!! How will be  A)  Turmeric derived curcumin in the treatment of endometriosis with phytoetrogens, than Cabergolin. B) Statins have been studied far better with fewer side effects comparing Cabergolin

Follicular cyst How to follow her up??

How best to follow up if U visualize a cyst in vary ? The approach varies according to her age.  The Society of Radiologists in Ultrasound made in 2019 the following recommendations regarding reporting of simple adnexal cysts of suspected ovarian origin based on size and menopausal status :

·         A) premenopausal women

o    1) ≤3 cm: no need to report; if described, consider calling a "follicle" rather than a "cyst" to reduce patient anxiety

§  impression: normal ovaries/adnexa

§  recommendation: no follow-up

o    2) >3 to ≤5 cm: report presence of simple cyst(s) and largest cyst diameter

§  impression: benign finding in the physiologic size range

§  recommendation: no follow-up

o    3) >5 cm: report with all cyst diameters

§  impression: benign simple cyst

§  recommendation:

§  4) >5 to ≤7 cm: follow-up either in 2-6 months for resolution/re-characterization or in 6-12 months for growth rate assessment, but no follow up is needed if the cyst is exceptionally well-visualized/characterized and documented with confidence by the imager

§  5) >7 cm: follow-up either in 2-6 months for resolution/re-characterization or in 6-12 months for growth rate assessment

o    follow-up of cyst (previously >5 cm): describe in report with all cyst diameters if not resolved

§  Follow up report  possibilities :-1:-decreased in size

§  impression: benign inconsequential finding; decrease in size excludes neoplasm

§  recommendation: no further follow-up needed

§  Follow up report  possibilities :-2 -similar in size

§  impression: benign simple cyst with stability over ≥12 months, most likely nonneoplastic or very slow growing benign neoplasm

§  recommendation: follow-up at 2 years from initial study to document stability and understand growth rate

§  Follow up report  possibilities :-3 -increased in size

§  impression: enlarging simple cyst, most likely a benign neoplasm

§  recommendation: follow-up in 1 year to evaluate any further changes in size

·         Follow up report  possibilities :-in postmeno Type one behaviour 1:-postmenopausal women

o    ≤1 cm: no need to report

§  impression: normal ovaries/adnexa

§  recommendation: no follow-up

·         Follow up report  possibilities :-in postmeno Type one behaviour 2:-postmenopausal women

o    >1 to ≤3 cm: report presence of simple cyst(s) and largest cyst diameter

§  impression: benign inconsequential finding

§  recommendation: no follow-up

·         Follow up report  possibilities :-in postmeno Type one behaviour 3-postmenopausal women

o    >3 cm: report with all cyst diameters

§  impression: benign simple cyst

§  recommendation:

·         Follow up report  possibilities :-in postmeno Type one behaviour 4-postmenopausal women

§  >3 to ≤5 cm: follow-up either in 3-6 months for resolution/re-characterization or in 6-12 months for growth rate assessment, but no follow up is needed if the cyst is exceptionally well-visualized/characterized and documented with confidence by the imager

·         Follow up report  possibilities :-in postmeno Type one behaviour 5-postmenopausal women

§  >5 cm: follow-up either in 3-6 months for resolution/re-characterization or in 6-12 months for growth rate assessment

·         Follow up report  possibilities :-in postmeno Type one behaviour  6  ;postmenopausal women

o    follow-up of cyst (previously >3 cm): describe in report with all largest cyst diameters if not resolved

§  decreased in size

§  impression: benign simple cyst; decrease in size excludes neoplasm

§  recommendation: no further follow-up needed

·         Follow up report  possibilities :-in postmeno Type one behaviour 7postmenopausal women

§  similar in size

§  impression: benign simple cyst

§  recommendation: follow-up at 2 years from initial study to document stability

§  increased in size

§  impression: enlarging simple cyst, most likely a benign neoplasm

§  recommendation: follow-up in 1 year to evaluate any further changes in size

Note that these guidelines do not apply to hemorrhagic ovarian cysts.

Treatment and prognosis

·         large (>3 cm) or symptomatic cysts may undergo surgical resection

·         smaller asymptomatic cysts are treated conservatively

·         risk of malignancy in septated ovarian cysts with no papillary projections or solid components are also considered low and are usually followed up on ultrasound